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- Bacterial cellulose: a versatile biopolymer for wound dressing applicationsPublication . Portela, Raquel; R. Leal, Catarina; Almeida, Pedro L.; Sobral, RitaAlthough several therapeutic approaches are available for wound and burn treatment and much progress has been made in this area, room for improvement still exists, driven by the urgent need of better strategies to accelerate wound healing and recovery, mostly for cases of severe burned patients. Bacterial cellulose (BC) is a biopolymer produced by bacteria with several advantages over vegetal cellulose, such as purity, high porosity, permeability to liquid and gases, elevated water uptake capacity and mechanical robustness. Besides its biocompatibility, BC can be modified in order to acquire antibacterial response and possible local drug delivery features. Due to its intrinsic versatility, BC is the perfect example of a biotechnological response to a clinical problem. In this review, we assess the BC main features and emphasis is given to a specific biomedical application: wound dressings. The production process and the physical-chemical properties that entitle this material to be used as wound dressing namely for burn healing are highlighted. An overview of the most common BC composites and their enhanced properties, in particular physical and biological, is provided, including the different production processes. A particular focus is given to the biochemistry and genetic manipulation of BC. A summary of the current marketed BC-based wound dressing products is presented, and finally, future perspectives for the usage of BC as wound dressing are foreseen.
- Cell necklaces behave as a soft glassy materialPublication . Franco, Jose M.; Patricio, Pedro; Almeida, Pedro L.; Portela, Raquel; Sobral, Rita; R. Leal, CatarinaPreviously we have reported a complex and rich viscoelastic behavior observed during the Planktonic growth of S. aureus strain COL1. In particular, in stationary shear flow, the viscosity Keeps increasing during the exponential phase and returns close to its initial value for the late phase of growth, where the bactéria population stabilizes. In oscillatory flow, the elastic and viscous moduli exhibit power--‐law behaviors whose exponentes are dependente on the bacteria growth stage. These power--‐law dependencies of G’ and G’’ match a Soft Glassy Material behavior. To describe this observed behavior, we have hypothesized a microscopic model considering the formation of a dynamic web--‐like structure, where percolation phenomena can occur, depending on growth stage and cell density. In this communication we describe the formation of these web--‐like structures, resembling cell necklaces at a specific time interval during bacterial growth. These findings were obtained by combining the previous data with new measurements performed in a rheometer with real--‐time image acquisition.
- Bacterial growth screened by rheologyPublication . Portela, Raquel; Pereira, M.; Sobral, Rita; Almeida, Pedro L.; R. Leal, CatarinaThe study of bacterial growth is a challenging field since it aims to describe the behaviour of microorganisms under different physical or chemical conditions. Bacterial growth as a biofilm is of particular interest as these gel-like structures are detrimental for all applications where “clean” surfaces are most important, and are related to failure of infection treatment, food spoilage and oil pipeline contamination, amongst others. In the literature one can find several works concerning the characterization of the mechanical behaviour of bacterial biofilms, although mostly are implemented over solid biofilms, as they appear in real situations [1-3], to study the adhesion properties in surfaces. In this work we applied a different technique to monitor the growth rate of a coccoid shaped bacterial species, the human pathogen Staphylococcus aureus.
- S. aureus and E. coli Co-culture Growth Under ShearPublication . Portela, Raquel; Almeida, Pedro L.; Sobral, Rita; R. Leal, CatarinaGrowing monocultures of two different species of human commensal/pathogenic bacteria, Staphylococcus aureus – a non-motile grampositive coccus and Escherichia coli – a motile gram-negative rod, were characterized using a real-time in situ rheology and rheo-imaging strategy. Subjecting bacterial populations to a shear flow is a closer approximation to bacterial thriving in the host, where they experience mechanical forces such as arterial or venous pressure. For both cultures, as the cell density of the population increases, cells rearrange themselves in different aggregates, capable of strongly influencing their environment, and leading to very different physical rheological responses, where motility appears to be determinant. One of the most striking observations is the behavior of the viscosity growth curve, showing dramatic value variations, with no counterpart in traditional biological measurements, as well as the coupling between translational and rotational motion of the E. coli aggregates along the growth curve [1], while S. aureus cells tend to sediment [2], over long periods of time. In the present study, a similar approach was applied to a co-culture of these two bacteria, S. aureus and E. coli, to evaluate the effect of possible interspecies interactions on the viscosity curve of the culture, during growth, when subject to a shear flow. Surprisingly, the observed behavior of the viscosity growth curve was enhanced in comparison to each individual curve and reveals a combination of details specific of each monoculture, suggesting synergy between these two bacterial species. After the rheological analysis, the final co-culture was recovered and inoculated on different solid media that allow to distinguish the development of S. aureus or E. coli colonies. Unexpectedly, S. aureus showed the capacity to accelerate its growth rate relatively to E. coli, when the two-species community is subjected to a shear flow. This behavior may reflect the occurrence of specific growth adaptations during co-culture upon shear flow, getting one step closer to physiological conditions.
- Crosslinked bacterial cellulose hydrogels for biomedical applicationsPublication . Almeida, Ana; Saraiva, João N.; Cavaco, Gonçalo; Portela, Raquel; Leal, Catarina R.; Sobral, Rita; Almeida, Pedro L.The skin, fundamental barrier that protects internal tissues, prevents pathogen invasion, and maintains the body fluid equilibrium, may be compromised upon traumas, such as incisions and burns. The healing process of such wounds is costly and usually hindered by the patient’s physiological conditions, associated diseases, inflammation and external factors, namely bacterial infections. Recently, increasing attention has been given to bacterial cellulose-based membranes to be applied as dressings for healing purposes. Bacterial cellulose is an attractive biomaterial due to its unique structural characteristics such as high porosity, high water retention capacity, high mechanical strength, low density, and biodegradability. One drawback of bacterial cellulose hydrogels is that, after the first dehydration, the water retention capacity is hindered. In this work we produced, modified, and characterized hydrated and de-hydrated BC membranes. Two crosslinking methods were adopted (using citric acid and epichlorohydrin as crosslinking agents), and the results obtained from the characterizations such as water retention capacity, mechanical properties or contact angle were compared to those of unmodified bacterial cellulose. We demonstrate that the cross-linked bacterial cellulose membranes present physical properties suitable to be used as surgical and burn wound dressings when hydrated, or as exuding wound dressings, diapers dressing or sanitary pads when dehydrated.
- Staphylococcus aureus strains rheology during growthPublication . Portela, Raquel; Almeida, Pedro L.; Patricio, Pedro; M.T., Cidade; Sobral, Rita; R. Leal, CatarinaThe study of the mechanical properties of living bacteria in a liquid rich medium, environment commonly used in laboratorial settings, opens a new perspective on the bacterial physiology and behaviour during population growth. In this work, the human pathogen Staphylococcus aureus was used as a study model due to its coccoid shape and regular morphology: MRSA strain COL [1] and its mutant strain RUSAL9 [2], which presents a deficient daughter-cell separation mechanism. Cultures were grown under a mechanical stress solicitation in parallel with optical density monitorization. Complex viscoelastic behaviour was revealed by these bacterial systems [3]. In particular the shear viscosity measurement during growth time, for a constant shear rate, showed an unexpected behaviour that cannot be observed by common optical experimental techniques.
- Antibiotic activity screened by the rheology of S. aureus culturesPublication . Portela, Raquel; Valcovo, Filipe; Almeida, Pedro L.; Sobral, Rita; Leal, Catarina R.Multidrug resistant bacteria are one of the most serious public health threats nowadays. How bacteria, as a population, react to the presence of antibiotics is of major importance to the outcome of the chosen treatment. In this study we addressed the impact of oxacillin, a β-lactam, the most clinically relevant class of antibiotics, in the viscosity profile of the methicillin resistant Staphylococcus aureus (MRSA) strain COL. In the first approach, the antibiotic was added, at concentrations under the minimum inhibitory concentration (sub-MIC), to the culture of S. aureus and steady-state shear flow curves were obtained for discrete time points during the bacterial growth, with and without the presence of the antibiotic, showing distinct viscosity progress over time. The different behaviors obtained led us to test the impact of the sub-inhibitory concentration and a concentration that inhibited growth. In the second approach, the viscosity growth curves were measured at a constant shear rate of 10 s−1, over time. The obtained rheological behaviors revealed distinctive characteristics associated to the presence of each concentration of the tested antibiotic. These results bring new insights to the bacteria response to a well-known bacteriolytic antibiotic.
- Motility and cell shape roles in the rheology of growing bacteria culturesPublication . Portela, Raquel; Almeida, Pedro L.; Sobral, Rita; R. Leal, CatarinaCell shape, size and self-motility appear as determinant intrinsic cell factors in the rheological behavior of living bacterial cultures during the growth process. In this work three different species were considered due to their differences on these intrinsic characteristics: two different strains of Staphylococcus aureus – strain COL and its isogenic cell wall autolysis mutant, RUSAL9 – both non-motile and Escherichia coli and Bacillus subtilis – both presenting intrinsic motility. In situ real-time rheology, was used to characterize the activity of growing bacteria, under steady-shear conditions, in particular the viscosity growth curve was measured, for a constant shear flow rate, presenting for all studied cultures, different and rich flow curves. These complex rheological behaviors are a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties, where cell size and shape and intrinsic motility are major players.