Luciana Gomes

Escherichia coli adhesion, biofilm development and antibiotic susceptibility on biomedical materials

The aim of this work was to test materials typically used in the construction of medical devices regarding their influence in the initial adhesion, biofilm development and antibiotic susceptibility of Escherichia coli biofilms. Adhesion and biofilm development was monitored in 12-well microtiter plates containing coupons of different biomedical materials--silicone (SIL), stainless steel (SS) and polyvinyl chloride (PVC)--and glass (GLA) as control. The susceptibility of biofilms to ciprofloxacin and ampicillin was assessed, and the antibiotic effect in cell morphology was observed by scanning electron microscopy. The surface hydrophobicity of the bacterial strain and materials was also evaluated from contact angle measurements. Surface hydrophobicity was related with initial E. coli adhesion and subsequent biofilm development. Hydrophobic materials, such as SIL, SS, and PVC, showed higher bacterial colonization than the hydrophilic GLA. Silicone was the surface with the greatest number of adhered cells and the biofilms formed on this material were also less susceptible to both antibiotics. It was found that different antibiotics induced different levels of elongation on E. coli sessile cells. Results revealed that, by affecting the initial adhesion, the surface properties of a given material can modulate biofilm buildup and interfere with the outcome of antimicrobial therapy. These findings raise the possibility of fine-tuning surface properties as a strategy to reach higher therapeutic efficacy.

96-well microtiter plates for biofouling simulation in biomedical settings

Microtiter plates with 96 wells are routinely used in biofilm research mainly because they enable high-throughput assays. These platforms are used in a variety of conditions ranging from static to dynamic operation using different shaking frequencies and orbital diameters. The main goals of this work were to assess the influence of nutrient concentration and flow conditions on biofilm formation by Escherichia coli in microtiter plates and to define the operational conditions to be used in order to simulate relevant biomedical scenarios. Assays were performed in static mode and in incubators with distinct orbital diameters using different concentrations of glucose, peptone and yeast extract. Computational fluid dynamics (CFD) was used to simulate the flow inside the wells for shaking frequencies ranging from 50 to 200 rpm and orbital diameters from 25 to 100 mm. Higher glucose concentrations enhanced adhesion of E. coli in the first 24 h, but variation in peptone and yeast extract concentration had no significant impact on biofilm formation. Numerical simulations indicate that 96-well microtiter plates can be used to simulate a variety of biomedical scenarios if the operating conditions are carefully set.

Macroscale versus microscale methods for physiological analysis of biofilms formed in 96-well microtiter plates

Microtiter plates with 96 wells have become one of the preferred platforms for biofilm studies mainly because they enable high-throughput assays. In this work, macroscale and microscale methods were used to study the impact of hydrodynamic conditions on the physiology and location of Escherichia coli JM109(DE3) biofilms formed in microtiter plates. Biofilms were formed in shaking and static conditions, and two macroscale parameters were assayed: the total amount of biofilm was measured by the crystal violet assay and the metabolic activity was determined by the resazurin assay. From the macroscale point of view, there were no statistically significant differences between the biofilms formed in static and shaking conditions. However, at a microscale level, the differences between both conditions were revealed using scanning electron microscopy (SEM). It was observed that biofilm morphology and spatial distribution along the wall were different in these conditions. Simulation of the hydrodynamic conditions inside the wells at a microscale was performed by computational fluid dynamics (CFD). These simulations showed that the shear strain rate was unevenly distributed on the walls during shaking conditions and that regions of higher shear strain rate were obtained closer to the air/liquid interface. Additionally, it was shown that wall regions subjected to higher shear strain rates were associated with the formation of biofilms containing cells of smaller size. Conversely, regions with lower shear strain rate were prone to have a more uniform spatial distribution of adhered cells of larger size. The results presented on this work highlight the wealth of information that may be gathered by complementing macroscale approaches with a microscale analysis of the experiments.

Effects of antibiotic concentration and nutrient medium composition on Escherichia coli biofilm formation and green fluorescent protein expression

Abstract Recombinant protein production processes have to maximise yield while minimising cost, which involves balancing plasmid maintenance with cell growth and protein expression. The aim of this study was to analyse the influence of two factors on heterologous protein production in Escherichia coli biofilm cells—the concentration of antibiotic used to maintain the selective pressure and the nutrient medium composition. Escherichia coli JM109(DE3) cells transformed with plasmid pFM23 for enhanced green fluorescent protein (eGFP) expression and containing a kanamycin resistance gene were used. They were exposed to 20 or 30 &mgr;g mL−1 kanamycin during biofilm growth in two different culture media, a diluted medium (DM) or the lysogeny broth (LB). The higher antibiotic concentration increased the specific eGFP production in planktonic cells, whereas no increase was detected in biofilm cells. Biofilm formation was increased in DM when compared to LB. Nevertheless, bacteria grown in LB had higher eGFP production than those grown in DM in both planktonic and sessile states (20‐fold and 2‐fold, respectively). Therefore, among the conditions tested, LB supplemented with 20 &mgr;g mL−1 kanamycin was the most advantageous medium to obtain the highest specific eGFP production in biofilm cells.

Biofilm Localization in the Vertical Wall of Shaking 96-Well Plates

Microtiter plates with 96 wells are being increasingly used for biofilm studies due to their high throughput, low cost, easy handling, and easy application of several analytical methods to evaluate different biofilm parameters. These methods provide bulk information about the biofilm formed in each well but lack in detail, namely, regarding the spatial location of the biofilms. This location can be obtained by microscopy observation using optical and electron microscopes, but these techniques have lower throughput and higher cost and are subjected to equipment availability. This work describes a differential crystal violet (CV) staining method that enabled the determination of the spatial location of Escherichia coli biofilms formed in the vertical wall of shaking 96-well plates. It was shown that the biofilms were unevenly distributed on the wall with denser cell accumulation near the air-liquid interface. The results were corroborated by scanning electron microscopy and a correlation was found between biofilm accumulation and the wall shear strain rates determined by computational fluid dynamics. The developed method is quicker and less expensive and has a higher throughput than the existing methods available for spatial location of biofilms in microtiter plates.

13C Metabolic Flux Analysis: From the Principle to Recent Applications

Metabolic flux analysis (MFA) has become a fundamental tool for quantifying metabolic pathways, which is essential for in-depth understanding of biological systems. In experimentally based MFA, isotopically labeled substrates are supplied to a biological system and the resulting labeling patterns are analyzed to obtain internal flux information. Three main techniques are necessary for 13 C MFA: (i) a steady state cell culture in a defined medium with 13 C substrates; (ii) precise measurements of the labeling pattern of targeted metabolites by nuclear magnetic resonance (NMR) or mass spectrometry (MS); (iii) mathematical modeling for experimental design, data processing and flux calculation. Recently, important technical advances have been made. The high costs of labeled substrates generate a demand for small cell cultivation volumes. The development of analytical instruments allows the measurement of 13 C enrichments with high accuracy and sensitivity. Moreover, powerful flux calculation algorithms have reduced computational efforts. Dynamic labeling experiments are also opening new possibilities for the investigation of specific pathways. While MFA is quite widely established in the study of microbial physiology, it is still a challenge to apply MFA to mammalian cells and plants. However, 13 C MFA techniques are continuously enhanced to better discern compartmentalized behaviors, which can help to characterize diseased metabolic states and improve metabolic engineering efforts in plants and other complex systems. The main objective of this work is to present the basic experimental and analytical methods of 13 C MFA, as well as representative examples of the latest approaches and findings of MFA in microorganisms, mammalian cells and plants.

Comparing the Recombinant Protein Production Potential of Planktonic and Biofilm Cells

Recombinant protein production in bacterial cells is commonly performed using planktonic cultures. However, the natural state for many bacteria is living in communities attached to surfaces forming biofilms. In this work, a flow cell system was used to compare the production of a model recombinant protein (enhanced green fluorescent protein, eGFP) between planktonic and biofilm cells. The fluorometric analysis revealed that when the system was in steady state, the average specific eGFP production from Escherichia coli biofilm cells was 10-fold higher than in planktonic cells. Additionally, epifluorescence microscopy was used to determine the percentage of eGFP-expressing cells in both planktonic and biofilm populations. In steady state, the percentage of planktonic-expressing cells oscillated around 5%, whereas for biofilms eGFP-expressing cells represented on average 21% of the total cell population. Therefore, the combination of fluorometric and microscopy data allowed us to conclude that E. coli biofilm cells can have a higher recombinant protein production capacity when compared to their planktonic counterparts.

SEM Analysis of Surface Impact on Biofilm Antibiotic Treatment

The aim of this work was to use scanning electron microscopy (SEM) to investigate the effect of ampicillin treatment on Escherichia coli biofilms formed on two surface materials with different properties, silicone (SIL) and glass (GLA). Epifluorescence microscopy (EM) was initially used to assess biofilm formation and killing efficiency on both surfaces. This technique showed that higher bacterial colonization was obtained in the hydrophobic SIL than in the hydrophilic GLA. It has also shown that higher biofilm inactivation was attained for GLA after the antibiotic treatment (7-log reduction versus 1-log reduction for SIL). Due to its high resolution and magnification, SEM enabled a more detailed analysis of the antibiotic effect on biofilm cells, complementing the killing efficiency information obtained by EM. SEM micrographs revealed that ampicillin-treated cells have an elongated form when compared to untreated cells. Additionally, it has shown that different materials induced different levels of elongation on cells exposed to antibiotic. Biofilms formed on GLA showed a 37% higher elongation than those formed on SIL. Importantly, cell elongation was related to viability since ampicillin had a higher bactericidal effect on GLA-formed biofilms. These findings raise the possibility of using SEM for understanding the efficacy of antimicrobial treatments by observation of biofilm morphology.

Impact of modified diamond-like carbon coatings on the spatial organization and disinfection of mixed-biofilms composed of Escherichia coli and Pantoea agglomerans industrial isolates

This work investigated the effects of diamond-like carbon (DLC) coatings on the architecture and biocide reactivity of dual-species biofilms mimicking food processing contaminants. Biofilms were grown using industrial isolates of Escherichia coli and Pantoea agglomerans on bare stainless steel (SST) and on two DLC surface coatings (a-C:H:Si:O designated by SICON® and a-C:H:Si designated by SICAN) in order to evaluate their antifouling activities. Quantification and spatial organization in single- and dual-species biofilms were examined by confocal laser scanning microscopy (CLSM) using a strain specific labelling procedure. Those assays revealed that the E. coli isolate exhibited a higher adhesion to the modified surfaces and a decreased susceptibility to disinfectant in presence of P. agglomerans than alone in axenic culture. While SICON® reduced the short-term growth of E. coli in axenic conditions, both DLC surfaces increased the E. coli colonization in presence of P. agglomerans. However, both modified surfaces triggered a significantly higher log reduction of E. coli cells within mixed-species biofilms, thus the use of SICON® and SICAN surfaces may be a good approach to facilitate the disinfection process in critical areas of food processing plants. This study presents a new illustration of the importance of interspecies interactions in surface-associated community functions, and of the need to evaluate the effectiveness of hygienic strategies with relevant multi-species consortia.

Surface conditioning with Escherichia coli cell wall components can reduce biofilm formation by decreasing initial adhesion

Bacterial adhesion and biofilm formation on food processing surfaces pose major risks to human health. Non-efficient cleaning of equipment surfaces and piping can act as a conditioning layer that affects the development of a new biofilm post-disinfection. We have previously shown that surface conditioning with cell extracts could reduce biofilm formation. In the present work, we hypothesized that E. coli cell wall components could be implicated in this phenomena and therefore mannose, myristic acid and palmitic acid were tested as conditioning agents. To evaluate the effect of surface conditioning and flow topology on biofilm formation, assays were performed in agitated 96-well microtiter plates and in a parallel plate flow chamber (PPFC), both operated at the same average wall shear stress (0.07 Pa) as determined by computational fluid dynamics (CFD). It was observed that when the 96-well microtiter plate and the PPFC were used to form biofilms at the same shear stress, similar results were obtained. This shows that the referred hydrodynamic feature may be a good scale-up parameter from high-throughput platforms to larger scale flow cell systems as the PPFC used in this study. Mannose did not have any effect on E. coli biofilm formation, but myristic and palmitic acid inhibited biofilm development by decreasing cell adhesion (in about 50%). These results support the idea that in food processing equipment where biofilm formation is not critical below a certain threshold, bacterial lysis and adsorption of cell components to the surface may reduce biofilm buildup and extend the operational time.