Cell adhesion in microchannel multiple constrictions - Evidence of mass transport limitations

Biofilm growth (fouling) in microdevices is a critical concern in several industrial, engineering and health applications, particularly in novel high-performance microdevices often designed with complex geometries, narrow regions and multiple headers. Unfortunately, on these devices, the regions with local high wall shear stresses (WSS) also show high local fouling rates. Several explanations have been put forward by the scientific community, including the effect of cell transport by Brownian motion on the adhesion rate. In this work, for the first time, both WSS and convection and Brownian diffusion effects on cell adhesion were evaluated along a microchannel with intercalate constriction and expansion zones designed to mimic the hydrodynamics of the human body and biomedical devices. Convection and Brownian diffusion effects were numerically studied using a steady-state convective-diffusion model (convection, diffusion and sedimentation). According to the numerical results, the convection and Brownian diffusion effects on cell adhesion are effectively more significant in regions with high WSS. Furthermore, a good agreement was observed between experimental and predicted local Sherwood numbers, particularly at the entrance and within the multiple constrictions. However, further mechanisms should be considered to accurately predict cell adhesion in the expansion zones. The described numerical approach can be used as a way to identify possible clogging zones in microchannels, and defining solutions, even before the construction of the prototype

Characterization and biofouling potential analysis of two cyanobacterial strains isolated from Cape Verde and Morocco

This work assesses the isolation, identification and characterization of two cyanobacterial strains isolated from Morocco and Cape Verde, as well as their biofilm-forming ability at two different surfaces, in a long-term assay under controlled hydrodynamic conditions. Cyanobacteria are new sources of value-added compounds but also ubiquitous and harmful microfoulers on marine biofouling. In this work, the isolation and identification of two cyanobacterial strains isolated from Cape Verde and Morocco, as well as their biofilm-forming ability on glass and Perspex under controlled hydrodynamic conditions, were performed. Phylogenetic analysis revealed that cyanobacterial strains isolated belong to Leptothoe and Jaaginema genera (Leptothoe sp. LEGE 181153 and Jaaginema sp. LEGE 191154). From quantitative and qualitative data of wet weight, chlorophyll a content and biofilm thickness obtained by optical coherence tomography, no significant differences were found in biofilms developed by the same cyanobacterial strain on different surfaces (glass and Perspex). However, the biofilm-forming potential of Leptothoe sp. LEGE 181153 proved to be higher compared with Jaaginema sp. LEGE 191154, particularly at the maturation stage of biofilm development. Three-dimensional biofilm images obtained from confocal laser scanning microscopy showed different patterns between both cyanobacterial strains and also among the two surfaces. Because standard methodologies to evaluate cyanobacterial biofilm formation, as well as two different optical imaging techniques, were used, this work also highlights the possibility of integrating different techniques to evaluate a complex phenomenon like cyanobacterial biofilm development

A comparison of vegetable leaves and replicated biomimetic surfaces on the binding of Escherichia coli and Listeria monocytogenes

Biofouling in the food industry is a huge issue, and one possible way to reduce surface fouling is to understand how naturally cleaning surfaces based on biomimetic designs influence bacterial binding. Four self-cleaning leaves (Tenderheart cabbage, Cauliflower, White cabbage and Leek) were analysed for their surface properties and artificial re-plicates were produced. The leaves and surfaces were subjected to attachment, adhesion and retention assays using Escherichia coli and Listeria monocytogenes. For the attachment assays, the lowest cell numbers occurred on the least hydrophobic and smooth surfaces but were higher than the flat control surface, regardless of the strain. Following the ad-hesion assays, using L. monocytogenes, the Tenderheart and Cauliflower biomimetic re-plicated leaves resulted in significantly lowered cell adhesion. Following the retention assays, White cabbage demonstrated lower cell retention for both types of bacteria on the biomimetic replicated surface compared to the flat control surface. The biomimetic sur-faces were also more efficient at avoiding bacterial retention than natural leaves, with reductions of about 1 and 2 Log in L. monocytogenes and E. coli retention, respectively, on most of the produced surfaces. Although the surfaces were promising in reducing bac-terial binding, the results suggested that different experimental assays exerted different influences on the conclusions. This work demonstrated that consideration needs to be given to the environmental factors where the surface is to be used and that bacterial species influence the propensity of biofouling on a surface. (c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Institution of Chemical Engineers. This is an open access article under the CC BY license (http://creative-commons.org/licenses/by/4.0/)

An in vitro dynamic model of catheter-associated urinary tract infections to investigate the role of uncommon bacteria on the Escherichia coli microbial consortium

About 9% of nosocomial infections are attributed to catheter-associated urinary tract infections (CAUTIs). Uncommon bacteria (Delftia tusurhatensis) have been isolated in CAUTIs in combination with wellestablished pathogenic bacteria such as E. coli. Nonetheless, the reason why E. coli coexists with other bacteria instead of outcompeting and completely eliminating them are unknown. As such, a flow cell reactor simulating the hydrodynamic conditions found in CAUTIs (shear rate of 15 s-1) was used to characterize the microbial physiology of E. coli and D. tsuruhatensis individually and in consortium, in terms of growth kinetics and substrate uptake. Single-species biofilms showed that up to 48 h the CFU counts significantly increased for both species (p<0.05). After 48 h, both species stabilized with similar CFU values reaching log 6.24 CFU.cm2 for E. coli and log 6.31 CFU.cm2 for D. tsuruhatensis (p>0.05). The assessment of spatial distribution of dual-species biofilms by LNA/2´OMe-FISH revealed that E. coli and D. tsuruhatensis coexist and tend to co-aggregate over time, which implies that bacteria are able to cooperate synergistically. Substrate uptake measurements revealed that in artificial urine medium the bacteria metabolized lactic acid, uric acid (E. coli and D. tsuruhatensis) and citric acid (D. tsuruhatensis). In the consortium, D. tsuruhatensis consumed citric acid more rapidly, presumably leaving more uric acid available in the medium to be used by E. coli. In conclusion, metabolic cooperation between E. coli and uncommon species seems to occur when these species share the same environment, leading to the formation of a stable microbial community

Unveiling the Antifouling Performance of Different Marine Surfaces and Their Effect on the Development and Structure of Cyanobacterial Biofilms

Since biofilm formation by microfoulers significantly contributes to the fouling process, it is important to evaluate the performance of marine surfaces to prevent biofilm formation, as well as understand their interactions with microfoulers and how these affect biofilm development and structure. In this study, the long-term performance of five surface materials-glass, perspex, polystyrene, epoxy-coated glass, and a silicone hydrogel coating-in inhibiting biofilm formation by cyanobacteria was evaluated. For this purpose, cyanobacterial biofilms were developed under controlled hydrodynamic conditions typically found in marine environments, and the biofilm cell number, wet weight, chlorophyll a content, and biofilm thickness and structure were assessed after 49 days. In order to obtain more insight into the effect of surface properties on biofilm formation, they were characterized concerning their hydrophobicity and roughness. Results demonstrated that silicone hydrogel surfaces were effective in inhibiting cyanobacterial biofilm formation. In fact, biofilms formed on these surfaces showed a lower number of biofilm cells, chlorophyll a content, biofilm thickness, and percentage and size of biofilm empty spaces compared to remaining surfaces. Additionally, our results demonstrated that the surface properties, together with the features of the fouling microorganisms, have a considerable impact on marine biofouling potential

Cooperation or conflict? Impact of intraspecific diversity on Escherichia coli biofilms

Intraspecific diversity in biofilm communities is associated with enhanced survival and growth of the individual biofilm populations. In here, we assess if this apparent cooperative behavior still holds as the number of different strains in a biofilm increases. Using E. coli as a model organism, the influence of intraspecific diversity in biofilm populations composed of up to six different E. coli strains, was assessed. Biofilm quantification was evaluated by crystal violet (CV) staining and colony forming units (CFU) counts. In general, with the increasing number of strains in a biofilm, an increase in cell counts and a decrease in matrix production was observed. This observation was confirmed by cluster analysis that indicated that after 24h of biofilm formation the best model, according to the Bayesian information criterion (BIC), consisted of three clusters that grouped together biofilms with an equal number of strains. It hence appears that increased genotypic diversity in a biofilm leads E. coli to maximize the production of its offspring, in detriment of the production of public goods (i.e. matrix components), that would be beneficial to all strains individually and the consortium as a whole. Apart from the ecological implications, these results can be explored in the area of clinical biofilms, as a decrease in matrix production might render these intraspecies biofilms more sensitive to antimicrobial agents

Increased intraspecies diversity in Escherichia coli biofilms promotes cellular growth at the expense of matrix production

Intraspecies diversity in biofilm communities is associated with enhanced survival and growth of the individual biofilm populations. Studies on the subject are scarce, namely, when more than three strains are present. Hence, in this study, the influence of intraspecies diversity in biofilm populations composed of up to six different Escherichia coli strains isolated from urine was evaluated in conditions mimicking the ones observed in urinary tract infections and catheter-associated urinary tract infections. In general, with the increasing number of strains in a biofilm, an increase in cell cultivability and a decrease in matrix production were observed. For instance, single-strain biofilms produced an average of 73.1 µg·cm−2 of extracellular polymeric substances (EPS), while six strains biofilms produced 19.9 µg·cm−2. Hence, it appears that increased genotypic diversity in a biofilm leads E. coli to direct energy towards the production of its offspring, in detriment of the production of public goods (i.e., matrix components). Apart from ecological implications, these results can be explored as another strategy to reduce the biofilm burden, as a decrease in EPS matrix production may render these intraspecies biofilms more sensitive to antimicrobial agents.This work was financially supported by Base Funding—UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE—funded by national funds through the FCT/MCTES (PIDDAC); Project POCI-01-0145-FEDER-030431 (CLASInVivo) and project POCI-01-0145-FEDER-029841 (POLY-PREVENTT), funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; Strategic funding of UIDB/04469/2020 of the Centre of Biological Engineering–CEB–funded by national funds through the FCT; Project BeMundus Brazil Europe/Erasmus Mundus scholarship granted by BM13DF0014.info:eu-repo/semantics/publishedVersio

A synthetic biology approach to engineer "therapeutic" bacteria

The high incidence and mortality of solid tumors like breast cancer makes the development of novel therapeutic agents a high priority. Curcumin, a natural substance from the rhizome of Curcuma longa, has captured the attention of the scientific community. Pre-clinical trials and extensive research has demonstrated its ability to prevent cancer. Indeed, curcumin has been shown to target critical genes involved in angiogenesis, apoptosis, cell cycle and metastasis, and consequently to inhibit cell growth. Currently, the clinical use of curcumin is mainly limited by its poor bioavailability which implies repetitive oral doses in order to achieve the therapeutic concentrations inside the cell. The idea of the present work is to design a strategy that could link the common technique used to treat solid tumors (ultrasound) with the therapeutic effects of curcumin. The plan is to use the temperature increase (consequence of ultrasound treatment) to trigger the in situ expression of curcumin by engineered bacteria. Escherichia coli was chosen as the model organism in which the genes involved in the curcumin pathway will be cloned. Those genes (4-coumarate: CoA ligase, diketide-CoA synthase and curcumin synthase) were successfully cloned under the control of a temperature sensitive promoter (dnaK). The proof-of-concept that the dnaK promoter can be induced by a temperature increase, leading to the expression of the 3 necessary genes, is currently being tested, using several biochemical assays. Moreover, several knockouts (KO) of specific genes from the E. coli K-12 MG1655 genome were performed in order to maximize the production of curcumin. The deletion strategy, as well as the definition of the non-essential genes to be KO, was determined in silico. This strategy included one single KO (gnd gene) and the multiple KO of five non-essential genes for aerobic growth (fumA, fumB, fumC, ccmA and argO) and serA gene for anaerobic growth. After optimizing the genes expression under the control of the temperature inducible promoter, the several KO will be transformed with this construction to confirm the improvement of curcumin production

Production and Characterization of Graphene Oxide Surfaces against Uropathogens

Graphene and its functionalized derivatives have been increasingly applied in the biomedi-cal field, particularly in the production of antimicrobial and anti-adhesive surfaces. This study aimed to evaluate the performance of graphene oxide (GO)/polydimethylsiloxane (PDMS) composites against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. GO/PDMS composites containing different GO loadings (1, 3, and 5 wt.%) were synthesized and characterized regarding their morphol-ogy, roughness, and hydrophobicity, and tested for their ability to inhibit biofilm formation under conditions that mimic urinary tract environments. Biofilm formation was assessed by determining the number of total and culturable cells. Additionally, the antibacterial mechanisms of action of GO were investigated for the tested uropathogens. Results indicated that the surfaces containing GO had greater roughness and increased hydrophobicity than PDMS. Biofilm analysis showed that the 1 wt.% GO/PDMS composite was the most effective in reducing S. aureus biofilm formation. In oppo-sition, P. aeruginosa biofilms were not inhibited by any of the synthesized composites. Furthermore, 1% (w/v) GO increased the membrane permeability, metabolic activity, and endogenous reactive oxygen species (ROS) synthesis in S. aureus. Altogether, these results suggest that GO/PDMS com-posites are promising materials for application in urinary catheters, although further investigation is required

How do Graphene Composite Surfaces Affect the Development and Structure of Marine Cyanobacterial Biofilms?

The progress of nanotechnology has prompted the development of novel marine antifouling coatings. In this study, the influence of a pristine graphene nanoplatelet (GNP)-modified surface in cyanobacterial biofilm formation was evaluated over a long-term assay using an in vitro platform which mimics the hydrodynamic conditions that prevail in real marine environments. Surface characterization by Optical Profilometry and Scanning Electron Microscopy has shown that the main difference between GNP incorporated into a commercially used epoxy resin (GNP composite) and both control surfaces (glass and epoxy resin) was related to roughness and topography, where the GNP composite had a roughness value about 1000 times higher than control surfaces. The results showed that, after 7 weeks, the GNP composite reduced the biofilm wet weight (by 44%), biofilm thickness (by 54%), biovolume (by 82%), and surface coverage (by 64%) of cyanobacterial biofilms compared to the epoxy resin. Likewise, the GNP-modified surface delayed cyanobacterial biofilm development, modulated biofilm structure to a less porous arrangement over time, and showed a higher antifouling effect at the biofilm maturation stage. Overall, this nanocomposite seems to have the potential to be used as a long-term antifouling material in marine applications. Moreover, this multifactorial study was crucial to understanding the interactions between surface properties and cyanobacterial biofilm development and architecture over time