Maria Olívia Pereira

Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm.

A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented. The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope. The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume. Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.

Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review.

Pseudomonas aeruginosa is the most prevalent pathogen of cystic fibrosis (CF) lung disease. Its long persistence in CF airways is associated with sophisticated mechanisms of adaptation, including biofilm formation, resistance to antibiotics, hypermutability and customized pathogenicity in which virulence factors are expressed according the infection stage. CF adaptation is triggered by high selective pressure of inflamed CF lungs and by antibiotic treatments. Bacteria undergo genetic, phenotypic, and physiological variations that are fastened by the repeating interplay of mutation and selection. During CF infection development, P. aeruginosa gradually shifts from an acute virulent pathogen of early infection to a host-adapted pathogen of chronic infection. This paper reviews the most common changes undergone by P. aeruginosa at each stage of infection development in CF lungs. The comprehensive understanding of the adaptation process of P. aeruginosa may help to design more effective antimicrobial treatments and to identify new targets for future drugs to prevent the progression of infection to chronic stages.

Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces

Biomaterial-associated infections remain a serious concern in modern healthcare. The development of materials that can resist or prevent bacterial attachment constitutes a promising approach to dealing with this problem. Antimicrobial peptides (AMPs) and enzymes have been recognized as promising candidates for the new generation of antimicrobial surfaces. AMPs have been the focus of great interest in recent years owing to a low propensity for developing bacterial resistance, broad-spectrum activity, high efficacy at very low concentrations, target specificity, and synergistic action with classical antibiotics. Biofilm-dispersing enzymes have been shown to inhibit biofilm formation, detach established biofilm, and increase biofilm susceptibility to other antimicrobials. This review critically examines the potential of these protein-like compounds for developing antibacterial coatings by reporting their immobilization into different substrata using different immobilization strategies.

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

Abstract This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm2, and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.

New trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches

Antimicrobial peptides (AMPs) have a broad spectrum of activity and unspecific mechanisms of action. Therefore, they are seen as valid alternatives to overcome clinically relevant biofilms and reduce the chance of acquired resistance. This paper reviews AMPs and anti-biofilm AMP-based strategies and discusses ongoing and future work. Recent studies report successful AMP-based prophylactic and therapeutic strategies, several databases catalogue AMP information and analysis tools, and novel bioinformatics tools are supporting AMP discovery and design. However, most AMP studies are performed with planktonic cultures, and most studies on sessile cells test AMPs on growing rather than mature biofilms. Promising preliminary synergistic studies have to be consubstantiated and the study of functionalized coatings with AMPs must be further explored. Standardized operating protocols, to enforce the repeatability and reproducibility of AMP anti-biofilm tests, and automated means of screening and processing the ever-expanding literature are still missing.

Antibiotic resistance of mixed biofilms in cystic fibrosis: impact of emerging microorganisms on treatment of infection

Cystic fibrosis (CF) is a genetic disorder associated with multispecies infections where interactions between classical and newly identified bacteria might be crucial to understanding the persistent colonisation in CF lungs. This study investigated the interactions between two emerging species, Inquilinus limosus and Dolosigranulum pigrum, and the conventional CF pathogen Pseudomonas aeruginosa by evaluating the ability to develop biofilms of mixed populations and then studying their susceptibility patterns to eight different antimicrobials. Monospecies biofilms formed by I. limosus and D. pigrum produced significantly less biomass than P. aeruginosa and displayed greater sensitivity to antimicrobials. However, when in dual-species biofilms with P. aeruginosa, the emerging species I. limosus and D. pigrum were crucial in increasing tolerance of the overall consortia to most antibiotics, even without a change in the number of biofilm-encased cells. These results may suggest that revising these and other species interactions in CF might enable the development of more suitable and effective therapies in the future.

Casbane diterpene as a promising natural antimicrobial agent against biofilm-associated infections

Croton nepetaefolius is a native plant from northeastern Brazil that belongs to the Euphorbiaceae family. The biological action of this plant has been extensively explored, being the secondary metabolites responsible for its properties alkaloids, diterpenes, and triterpenes. This study aimed to evaluate the ability of casbane diterpene (CD), isolated from the ethanolic extract of C. nepetaefolius, to inhibit microbial growth and biofilm formation of several clinical relevant species (bacteria and yeasts). It was found that CD possessed biocidal and biostatic activity against the majority of the species screened, with minimal active concentrations ranging between 125 and 500 µg/mL. In addition, it was observed that biofilm formation was inhibited even when the planktonic growth was not significantly affected. In conclusion, CD showed potential to be a natural tool for the treatment of diseases caused by different infectious microorganisms.

Validation of respirometry as a short-term method to assess the efficacy of biocides

This study shows that a short-term respirometric measurement based on the rate of oxygen uptake needed to oxidize glucose is a reliable and fast method to assess biocide efficacy against P. fluorescens cells. Respiratory activity using oxygen consumption rate, the determination of viable and nonviable cells using Live/Dead® BacLight™ kit and colony formation units (CFU), were compared as indicators of the biocidal efficacy of ortho-phthalaldehyde (OPA). The results showed that determining the effect of OPA against P. fluorescens using the different methods leads to different conclusions. The minimum bactericidal concentration (MBC) was 80 mg l−1, 100 mg l−1 and 65 mg l−1 respectively, using respiratory activity, viability using BacLight counts and culturability. The plate count method was shown to underestimate the biocidal action of OPA, whilst data from respirometry and viability using Live/Dead BacLight kit correlated strongly and were not statistically different when yellow cells were considered nonviable. Respirometry therefore represents an expeditious, non-destructive and accurate method to determine the antimicrobial action of biocides against aerobic heterotrophic bacteria.

A versatile reactor for continuous monitoring of biofilm properties in laboratory and industrial conditions

Aims: The understanding of the dynamics of surface microbial colonization with concomitant monitoring of biofilm formation requires the development of biofilm reactors that enable direct and real‐time evaluation under different hydrodynamic conditions.

Antagonism between Bacillus cereus and Pseudomonas fluorescens in planktonic systems and in biofilms

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steady-state biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.