Optical and spectroscopic methods for biofilm examination and monitoring

Optical and spectroscopic methods for biofilmexamination and monitoring are reviewed.Biofilm examination techniques includemicroscopic methods, coupled with imageanalysis and with oligonucleotide ribosomal RNAprobing methods (fluorescence in situhybridization). Microscopic examinationtechniques are especially advantageous inextracting biofilm structural and architecturalparameters, as well as structure-functionrelationships of the biofilm microbialpopulation. Spectroscopic techniques are ableto elicit biofilm chemical and metabolicpatterns, as well as biofilm activity. They areof outstanding importance for on-line,non-invasive biofilm monitoring, especiallywhen coupled with chemometric algorithms forspectra calibration and pattern recognition.The paper emphasises the importance of thecombination of novel and established analyticaltechniques, as well as their integration withpowerful computational methods for theautomation of biofilm monitoring.     

Textural fingerprints: a comprehensive descriptor for biofilm structure development

Automated tools to determine biofilm structure are necessary to interpret large time series of biofilm images. Image analysis based on the evaluation of Spatial Gray Level Dependence Matrices (SGLDM) enabled us to monitor biofilm structure development in response to external disturbances (i.e., periodic increases of wall shear stress) at a large scale (i.e., >1 mm). We applied our method to an experiment conducted in an annular reactor over a 10-week period. Six states of biofilm development were differentiated by their unique structure. Previous exposure to rapidly increased shear influenced the resulting biofilm structure after additional shear increases. In addition, on the scale of the biofilm images, the biofilm structure after a shear increase was spatially heterogeneous and resulted in spatially differentiated regrowth after detachment at different locations in the biofilm. SGLDM was developed further as an alternative to approaches based on image binarization as binarization leads to information loss for low-magnification and low-resolution images. During post-processing of image data, structural states of biofilm development were identified by K-means clustering and data display in Principal Component plots. Quantitatively selected representative images were used to illustrate the meaning of the clusters. Post-treatment of image data was essential for managing several thousands of raw biofilm images and therefore improved the usefulness of the image analysis.     

Flexible biofilm monitoring device

Biofilms and their analysis are increasingly attracting the attention of the scientific community due to the immense importance and impact of biofilms in various natural, technical and medical fields. For these purposes, an optimized and extended antibiofilm assay system based on the Calgary Biofilm Device (MBEC Assay® system) consisting of microtiter plate and PCR tubes was established. Its implementation was used to study the growth characteristics of the sessile phenotype of Pseudomonas fluorescens exposed to antimicrobial peptides. Inhibitory effects of an antimicrobial peptide on P. fluorescens biofilm formation could be determined at a concentration of 250 μg/ml (biofilm prevention concentration (BPC)) using the modified biofilm assay. Similarly, the biofilm bactericidal concentration (BBC) at 125 μg/ml and the minimum biofilm elimination concentration to remove 90% of the total biofilm mass (MBEC90) were measured at a concentration range of 15.625–1.95 μg/ml. In conclusion, this optimized system provides a highly variable, simple, and cost‐effective alternative to high‐throughput screening based on the Calgary Biofilm Device (CBD).     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

The influence of biofilm structure and total interaction energy on Escherichia coli retention by Pseudomonas aeruginosa biofilm

The retention of a surrogate pathogenic bacterium, Escherichia coli^T , in Pseudomonas aeruginosa biofilms (with various EPS excreting capacities) was investigated using a laboratory flow cell system. The structural characteristics of the biofilm, as well as the quantity of E. coli^T retained in the biofilm, were assessed using confocal laser scanning microscopy coupled with image analysis. In addition, the total interaction energy between E. coli^T and the P. aeruginosa biofilm was computed with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, which provided an additional context to explain the pathogen interaction in aquatic biofilms. The correlations between the quantity of detained E. coli^T cells and the structural characteristics of the biofilm were analysed and the results indicated that the heterogeneity of the biofilm could create a quiescent zone leading to temporary retention of E. coli^T within the biofilm. Overall, this study provided insights toward understanding the retention of pathogenic bacteria in environmental biofilms.     

Nitrifying activity monitoring and kinetic parameters determination in a biofilm airlift reactor by respirometry

The applicability of batch respirometry, as a simple technique for monitoring off-line nitrifying activity and kinetic parameters, was evaluated using two sets of ammonia and nitrite concentrations. The O₂ uptake rate (OUR) profiles obtained from the assays were adjusted to a substrate inhibition model. The maximum specific ammonia-oxidizing biomass activity (r_Smax) was 0.079 g N-NH₄ ⁺ g VSS^–1 d^–1 with a half saturation coefficient (K_S) of 11 mg N-NH₄ ⁺ l^–1 and an inhibition coefficient (K_i) of 3300 mg N-NH₄ ⁺ l^–1. Besides, the maximum specific value of nitrite-oxidizing activity was 0.082 g N-NO₂ ^– g VSS^–1 d^–1 with a K_S of 4.1 mg N-NO₂ ^– l^–1 and K_i of 1400 mg N-NO₂ ^– l^–1.     

Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Influence of membrane surface properties on the behavior of initial bacterial adhesion and biofilm development onto nanofiltration membranes

In order to investigate biofouling problems, the fundamental behaviors of initial bacterial adhesion and biofilm development on four different nanofiltration (NF) membranes were evaluated using Pseudomonas aeruginosa PAO1 as a model bacterial strain. Initial cell adhesion was considerably higher on an aromatic polyamide-based NF membrane with a hydrophobic and rough surface, whereas cell aggregation on a polypiperazine-based NF membrane with a relatively hydrophilic and smooth surface was lower. Moreover, significant differences in the structural heterogeneity of the biofilms were observed among the four NF membranes. This study shows that the surface roughness and hydrophobicity of a membrane play an important role in determining initial cell adhesion, aggregation and favorable localization sites for colony formation. In addition, it was found that biofilm development was strongly influenced by the surface morphology of a membrane.     

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.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

Reconstruction of biofilm images: combining local and global structural parameters

Digitized images can be used for quantitative comparison of biofilms grown under different conditions. Using biofilm image reconstruction, it was previously found that biofilms with a completely different look can have nearly identical structural parameters and that the most commonly utilized global structural parameters were not sufficient to uniquely define these biofilms. Here, additional local and global parameters are introduced to show that these parameters considerably increase the reliability of the image reconstruction process. Assessment using human evaluators indicated that the correct identification rate of the reconstructed images increased from 50% to 72% with the introduction of the new parameters into the reconstruction procedure. An expanded set of parameters especially improved the identification of biofilm structures with internal orientational features and of structures in which colony sizes and spatial locations varied. Hence, the newly introduced structural parameter sets helped to better classify the biofilms by incorporating finer local structural details into the reconstruction process.     

调节生物被膜化合物的研究进展

细菌的生物被膜是其在自然界中一种常见的生存状态。生物被膜的形成是细菌耐药性产生的主要机制之一,也是许多感染性疾病难以控制的重要原因。由于生物被膜在传染性疾病中的突出地位和细菌多重耐药性的蔓延,目前急需研制开发出能够调节生物被膜形成的新型抗菌药物。文中对调节生物被膜形成和发育的小分子抑制剂进行了详细的综述。     

In vitro biofilm model for studying tongue flora and malodour

AIMS: To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. METHODS AND RESULTS: Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/-SEM) growth rate for 72-h biofilms (n=4) was micro=0.014 h(-1) (+/-0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient>0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. CONCLUSIONS: The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.     

Three-dimensional biofilm image reconstruction for assessing structural parameters

Parameters representing three-dimensional (3D) biofilm structure are quantified from confocal laser-scanning microscope (CLSM) images. These 3D parameters describe the distribution of biomass pixels within the space occupied by a biofilm; however, they lack a direct connection to biofilm activity. As a result, researchers choose a handful of parameters without there being a consensus on a standard set of parameters. We hypothesized that a select 3D parameter set could be used to reconstruct a biofilm image and that the reconstructed and original biofilm images would have similar activities. To test this hypothesis, an algorithm was developed to reconstruct a biofilm image with parameters identical to those of the original CLSM image. We introduced an objective method to assess the reconstruction algorithm by comparing the activities of the original and reconstructed biofilm images. We found that biofilm images with identical structural parameters showed nearly identical activities and substrate concentration profiles. This implies that the set containing all common structural parameters can successfully describe biofilm structure. This finding is significant, as it opens the door to the next step, of finding a smaller standard set of biofilm structural parameters that can be used to compare biofilm structure.     

Development and characterization of a fiber optical fluorescence sensor for the online monitoring of biofilms and their microenvironment

The growth of microorganisms on surfaces and interfaces as a biofilm is very common and plays important role in various areas such as material science, biomedicine, or waste treatment among others. Due to their inhomogeneous structure and the variance in the microorganism consortium, the analysis of biofilms represents a significant challenge. An online fluorescence sensor was developed that is able to measure the most important biological fluorophores (proteins, nicotinamide adenine dinucleotide, and flavin) in a noninvasive manner in biofilms, e.g. in bioelectrochemical applications. The sensor gives the opportunity to continuously draw conclusions on the metabolic state of the biofilm. The developed sensor has a diameter of 1 mm at the sensor tip and can be moved on and into the biofilm surface. In the first experiment, the measuring range of the sensor and the long‐term stability could be determined and the system applicability was confirmed. In addition, measurements in biofilm‐like structures could be performed. The formation of a wastewater‐based biofilm was monitored using the developed sensor, demonstrating the functionality of the sensor in a proof‐of‐principle experiment.     

Methanotrophic attached-film reactor development and biofilm characteristics

The feasibility of using methanotrophs in an attached-film, fluidized-bed (MAFFB) reactor system has been under investigation since 1987. Mixed culture, methane-utilizing attached biofilms were developed on diatomaceous earth particles and on granular activated carbon. The required feed gases, methane and oxygen, were supplied to the attached biofilm in disolved form using separate gas-liquid aeration columns. Biofilm growth was steady despite low influent dissolved methane concentrations (1 to 3 mg/L). A breeder MAFFB operated consistently for 4.1 years with attached biofilm concentrations as high as 51.7 g VS/L static-bed with minimal biomass wasting and with minimal buffer and nutrient inputs. The maximum biomass concentration observed was 75.6 g VS/L static-bed in a MAFFB reactor treating trichloroethene. Biofilm thickness reached 160 mum with typical values of 70 mum under methane and oxygen growht-rate-limited conditions. Biofilm densities of 120 to 190 g VS/L film were observed. Growth rates varied from <0.01/d to 0.17/d. Greater than 90% of the biomass concentration in the bed was attached, and effluent total suspended solids ranged from 5 to 74 mg/L, with an average of 24 mg/L over 27 runs in four MAFFB systems at upflow velocities of 11.4 to 25 m/h. Heterotrophic attached-film methanotrophs appear to be stable and useful for applications in toxics treatment, and other product manipulations. (c) 1992 John Wiley & Sons, Inc.     

铜绿假单胞菌生物膜分散的研究近况

细菌分泌胞外多糖附着在物体表面组成一个结构性群体即生物膜,导致对抗生素的强抵抗性和感染的迁延不愈。反过来,已形成的生物膜也可以分散为游离菌,许多环境物质能够促进该分散过程,并且这些物质与抗生素合用对生物膜有强大的对抗作用。从生物膜到浮游菌是个复杂的过程,目前关于铜绿假单胞菌生物膜分散的特征、机制、诱导分子等已经引起了学者的强烈兴趣,随着问题的深入研究必然会给人类治疗生物膜所致的难治性感染带来更大的意义。