Filipe Mergulhão

Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow

Biofilm formation is a major factor in the growth and spread of both desirable and undesirable bacteria as well as in fouling and corrosion. In order to simulate biofilm formation in industrial settings a flow cell system coupled to a recirculating tank was used to study the effect of a high (550 mg glucose l−1) and a low (150 mg glucose l−1) nutrient concentration on the relative growth of planktonic and attached biofilm cells of Escherichia coli JM109(DE3). Biofilms were obtained under turbulent flow (a Reynolds number of 6000) and the hydrodynamic conditions of the flow cell were simulated by using computational fluid dynamics. Under these conditions, the flow cell was subjected to wall shear stresses of 0.6 Pa and an average flow velocity of 0.4 m s−1 was reached. The system was validated by studying flow development on the flow cell and the applicability of chemostat model assumptions. Full development of the flow was assessed by analysis of velocity profiles and by monitoring the maximum and average wall shear stresses. The validity of the chemostat model assumptions was performed through residence time analysis and identification of biofilm forming areas. These latter results were obtained through wall shear stress analysis of the system and also by assessment of the free energy of interaction between E. coli and the surfaces. The results show that when the system was fed with a high nutrient concentration, planktonic cell growth was favored. Additionally, the results confirm that biofilms adapt their architecture in order to cope with the hydrodynamic conditions and nutrient availability. These results suggest that until a certain thickness was reached nutrient availability dictated biofilm architecture but when that critical thickness was exceeded mechanical resistance to shear stress (ie biofilm cohesion) became more important.

Critical review on biofilm methods

Abstract Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Current and emergent strategies for disinfection of hospital environments

Abstract A significant number of hospital-acquired infections occur due to inefficient disinfection of hospital surfaces, instruments and rooms. The emergence and wide spread of multiresistant forms of several microorganisms has led to a situation where few compounds are able to inhibit or kill the infectious agents. Several strategies to disinfect both clinical equipment and the environment are available, often involving the use of antimicrobial chemicals. More recently, investigations into gas plasma, antimicrobial surfaces and vapour systems have gained interest as promising alternatives to conventional disinfectants. This review provides updated information on the current and emergent disinfection strategies for clinical environments.

The influence of nonconjugative Escherichia coli plasmids on biofilm formation and resistance

Aims:  This work describes the effects of the presence of nonconjugative plasmids in Escherichia coli cells forming biofilms on a flow cell system under turbulent conditions.

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.

The Influence of Interfering Substances on the Antimicrobial Activity of Selected Quaternary Ammonium Compounds

Standard cleaning processes may not remove all the soiling typically found in food industry, such as carbohydrates, fats, or proteins. Contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants. The influence of alginic acid, bovine serum albumin, yeast extract, and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against Bacillus cereus vegetative cells and Pseudomonas fluorescens. The bacteria (single and consortium) were exposed to surfactants (single and combined) in the absence and presence of potential disinfection interfering substances. The antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation. The tested surfactants were efficient against both bacteria (single and consortium) with minimum bactericidal concentrations ranging from 3 to 35 mg·L−1. The strongest effect was caused by humic acids that severely quenched antimicrobial action, increasing the minimum bactericidal concentration of the surfactants on P. fluorescens and the consortium. The inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity. This study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies.

The effects of surface properties on Escherichia coli adhesion are modulated by shear stress.

The adhesion of Escherichia coli to glass and polydimethylsiloxane (PDMS) at different flow rates (between 1 and 10 ml s(-1)) was monitored in a parallel plate flow chamber in order to understand the effect of surface properties and hydrodynamic conditions on adhesion. Computational fluid dynamics was used to assess the applicability of this flow chamber in the simulation of the hydrodynamics of relevant biomedical systems. Wall shear stresses between 0.005 and 0.07 Pa were obtained and these are similar to those found in the circulatory, reproductive and urinary systems. Results demonstrate that E. coli adhesion to hydrophobic PDMS and hydrophilic glass surfaces is modulated by shear stress with surface properties having a stronger effect at the lower and highest flow rates tested and with negligible effects at intermediate flow rates. These findings suggest that when expensive materials or coatings are selected to produce biomedical devices, this choice should take into account the physiological hydrodynamic conditions that will occur during the utilization of those devices.

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.

Setup and Validation of Flow Cell Systems for Biofouling Simulation in Industrial Settings

A biofouling simulation system consisting of a flow cell and a recirculation tank was used. The fluid circulates at a flow rate of 350 L· h−1 in a semicircular flow cell with hydraulic diameter of 18.3 mm, corresponding to an average velocity of 0.275 m· s−1. Using computational fluid dynamics for flow simulation, an average wall shear stress of 0.4 Pa was predicted. The validity of the numerical simulations was visually confirmed by inorganic deposit formation (using kaolin particles) and also by direct observation of pathlines of tracer PVC particles using streak photography. Furthermore, the validity of chemostat assumptions was verified by residence time analysis. The system was used to assess the influence of the dilution rate on biofilm formation by Escherichia coli JM109(DE3). Two dilution rates of 0.013 and 0.0043 h−1 were tested and the results show that the planktonic cell concentration is increased at the lower dilution rate and that no significant changes were detected on the amount of biofilm formed in both conditions.

Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ‐proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ‐proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5′‐untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ‐proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.