Carla Ferreira

Physiological changes induced by the quaternary ammonium compound benzyldimethyldodecylammonium chloride on Pseudomonas fluorescens

OBJECTIVES Antimicrobial resistance is a major public health concern, particularly in hospitals and other healthcare settings. For the rational design of disinfection strategies, it is of utmost importance to understand the mechanisms of action of antimicrobials. In this study, the mechanism of action of benzyldimethyldodecylammonium chloride (BDMDAC) was assessed against Pseudomonas fluorescens. METHODS The targets of antimicrobial action were studied using different bacterial physiological indices. The MIC, MBC, membrane permeabilization, intracellular potassium release, physico-chemical surface properties, surface charge, outer membrane protein (OMP) expression and morphological changes were assessed after BDMDAC exposure. RESULTS The MIC was found to be 20 mg/L and the MBC was 10 mg/L. BDMDAC led to a significant change in cell surface hydrophobicity and induced propidium iodide uptake. Such results suggest cytoplasmic membrane damage, corroborated by the release of intracellular potassium. The results obtained from the zeta potential measurement demonstrate a -31.2 mV value for untreated cells and -21.0 mV for cells at the MIC. Scanning electron microscopy revealed that cells treated with 20 mg/L were less bulky, and their membrane seemed to be rougher, wrinkled and deformed when compared with untreated cells. The overall bactericidal events occurred without detectable changes in OMP expression. CONCLUSIONS BDMDAC is an effective biocide against P. fluorescens. It binds by ionic and hydrophobic interactions to the cell membrane, causing changes in membrane properties and function, as manifested by phenomena such as cellular disruption and loss of membrane integrity with consequent leakage of essential intracellular constituents.

Biofouling control using microparticles carrying a biocide

This study presents a new technological approach to minimize the use of antimicrobial (AMB) agents and their deleterious effects, based on the principle of drug-delivery systems whereby the AMB chemicals are transported on microparticles. The efficacy of microparticles carrying the quaternary ammonium compound (QAC), benzyldimethyldodecyl ammonium chloride (BDMDAC), was assessed against Pseudomonas fluorescens in both the planktonic and the biofilm state. The microparticles were prepared using a layer-by-layer (LBL) self-assembly technique. Oppositely charged molecules of polyethyleneimine (PEI), sodium polystyrene sulfonate (PSS), and BDMDAC were assembled on polystyrene (PS) cores. BDMDAC-coated particles were observed by CryoSEM and their composition analyzed by X-ray microanalysis. Zeta potential measurements indicated that changes in surface charge were compatible with a BDMDAC/particle interaction. This biocidal carrier structure had significant stability, verified by the release of only 15% of the BDMDAC when immersed in water for 18 months. Biocidal carrier activity was evaluated by determining the survival ratio of P. fluorescens planktonic and biofilm cells after different exposure periods to BDMDAC-coated particles. Tests with biofilm cells were also performed with the free QAC. An efficient AMB effect (minimum bactericidal concentration) against suspended cells was found for a concentration of 9.2 mg l−1 of BDMDAC on coated particles after incubation for 30 min and 6.5 mg l−1 of BDMDAC on coated particles after 60 min. Exposure of biofilms to PS-PEI/PSS/BDMDAC (0.87 mg l−1) resulted in a decrease in viability of 60.5% and 66.5% of the total biofilm population for 30 and 60 min exposure times, respectively. Exposure for 60 min to 6.33 mg l−1 and 11.75 mg l−1 of BDMDAC in PS-PEI/PSS/BDMDAC particles promoted inactivation of 80.6% and 87.2% of the total population, respectively. The AMB effects obtained with the application of free BDMDAC were statistically similar to those promoted by the application of BDMDAC coated particles. The overall results indicate that this novel AMB strategy has potential for the control of microbial growth of planktonic cells and biofouling. Moreover, the technique allows the reuse of AMB molecules and consequently reduces the environmental risks associated with excessive use of AMB agents, thereby providing real benefits to public health.

Occurrence of virulence genes in multidrug-resistant Escherichia coli isolates from Iberian wolves (Canis lupus signatus) in Portugal

While much evidence supports the view that the total consumption of antimicrobials is the critical factor in selecting resistance, the possibility of resistant isolates and/or genes encoding resistance being transferred among different living communities has raised serious concerns. In the present study, Escherichia coli isolates recovered from faecal samples (n = 34) of Iberian wolves (Canis lupus signatus) were characterized for their antimicrobial drug susceptibility. Nearly two thirds of the isolates carried resistance to one or more antimicrobial drugs (in a panel of 19 antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. By screening a set of 20 multidrug-resistant E. coli for virulence genes, we found strains positive for cdt, chuA, cvaC, eaeA, paa and bfpA, which was the most common virulence trait. Phylogenetic analyses have shown that the majority of these E. coli strains fall into phylogenetic groups A and B1. In this study, the diversity of extended-spectrum β-lactamase-producing strains was expressed by both polymorphism of the pulsed-field gel electrophoresis patterns and the presence of various resistance and virulence genes profiles. Finding the specific implications of these multi-resistant bacteria (hosting several virulence factors) in wolf conservation is a challenging topic to be addressed in further investigations.

Influence of nanohydroxyapatite surface properties on Staphylococcus epidermidis biofilm formation

Nanohydroxyapatite (nanoHA), due to its chemical properties, has appeared as an exceptionally promising bioceramic to be used as bone regeneration material. Staphylococcus epidermidis have emerged as major nosocomial pathogens associated with infections of implanted medical devices. In this work, the purpose was to study the influence of the nanoHA surface characteristics on S. epidermidis RP62A biofilm formation. Therefore, two different initial inoculum concentrations (Ci) were used in order to check if these would affect the biofilm formed on the nanoHA surfaces. Biofilm formation was followed by the enumeration of cultivable cells and by scanning electron microscopy. Surface topography, contact angle, total surface area and porosimetry of the biomaterials were studied and correlated with the biofilm data. The surface of nanoHA sintered at 830℃ (nanoHA830) showed to be more resistant to S. epidermidis attachment and accumulation than that of nanoHA sintered at 1000℃ (nanoHA1000). The biofilm formed on nanoHA830 presented differences in terms of structure, surface coverage and EPS production when compared to the one formed on nanoHA1000 surface. It was observed that topography and surface area of nanoHA surfaces had influence on the bacterial attachment and accumulation. Ci influenced bacteria attachment and accumulation on nanoHA surfaces over time. The choice of the initial inoculum concentration was relevant proving to have an effect on the extent of adherence thus being a critical point for human health if these materials are used in implantable devices. This study showed that the initial inoculum concentration and surface material properties determine the rate of microbial attachment to substrata and consequently are related to biofilm-associated infections in biomaterials.

Biofilm Control With New Microparticles With Immobilized Biocide

Biofouling of heat exchangers, pipes, and valves isan unavoidable hazard in industrial plants. Operators have to face the problems of cleaning the fouled equipment, treating the wastewater from the discharge of antimicrobial cleaning agents (biocides, surfactants, etc.), and protecting the consumer from contamination risks. To tackle these problems, a targeted cleaning technique is here proposed that unites efficient removal of the biological deposits (specifically targeting the places where deposits grow) with low consumption of cleaning agents. The goal is to develop microparticles with functionalized surfaces that act as carriers of biocidal molecules, attach to biofilm surfaces, and deliver the biocide on the desired sites. This novel biofilm control technology may provide cost-effective, environmental- and health-friendly strategies that will improve operational efficacy. Previously, our group had performed studies with polystyrene (PS) microparticles functionalized with the biocide benzyldimethyldodecylammonium chloride (BDMDAC) against biofilms of Pseudomonas fluorescens. BDMDAC-functionalized microparticles were prepared using the layer-by-layer self-assembly (LBL) technique. Since PS particles are very expensive, our group tested calcium carbonate microparticles (CaCO3) (diameter: 3 μm), produced by the same LBL assembly technique. These microparticles have the advantage of being cheaper, porous, and highly abundant in nature. In this study, P. fluorescens biofilms were exposed to 6.33 mg/L and 11.75 mg/L of BDMDAC-coated CaCO3 microparticles for 60 min. This strategy promoted inactivation of 81.9% (6.33 mg/L) and 93.3 % (11.75 mg/L mg/L) of the total population. As a control, free BDMDAC was tested against P. fluorescens biofilms, demonstrating similar inactivation (p > 0.05) under the new strategy.

Surface physicochemical properties of selected single and mixed cultures of microalgae and cyanobacteria and their relationship with sedimentation kinetics

BackgroundMicroalgae are photosynthetic microorganisms presenting a diversity of biotechnological applications. However, microalgal cultivation systems are not energetically and economically feasible. Possible strategies that can be applied to improve the feasibility of microalgal production include biofouling control in photobioreactors, the use of attached growth systems and bioflocculation. These processes are ruled by surface physicochemical properties. Accordingly, the surface physicochemical properties of Chlorella vulgaris, Pseudokirchneriella subcapitata, Synechocystis salina and Microcystis aeruginosa were determined through contact angle and zeta potential measurements. Additionally, mixed cultures of the selected microorganisms were performed. Sedimentation kinetics of the studied cultures was also evaluated to understand how surface physicochemical properties influence microalgal recovery.ResultsAll studied microorganisms, except S. salina, presented a hydrophilic surface. The co-culture of S. salina with the other studied microorganisms resulted in a more hydrophobic algal suspension. Regarding zeta potential determinations, all studied suspensions presented a negatively charged surface (approximately -40.8 ± 4.4 mV). Sedimentation experiments have shown that all microalgal suspensions presented low microalgal recovery efficiencies. However, a negative linear relationship between microalgal removal percentage and free energy of hydrophobic interaction was obtained.ConclusionsThe evidence of a relationship between microalgal removal percentage and free energy of hydrophobic interaction demonstrates the importance of surface physicochemical properties on microalgal settling. However, the low recovery efficiencies achieved, as well as the high net zeta potential values determined, indicate that another factor to consider in microalgal settling is the ionic strength of the culture medium, which play an important role in suspensions’ stability.

Comparative stability and efficacy of selected chlorine-based biocides against Escherichia coli in planktonic and biofilm states

Microbial contamination is an unavoidable problem in industrial processes. Sodium hypochlorite (SH) is the most common biocide used for industrial disinfection. However, in view of the current societal concerns on environmental and public health aspects, there is a trend to reduce the use of this biocide as it can lead to the formation of organochlorinated carcinogenic compounds. In this work the efficacy of SH was assessed against Escherichia coli in planktonic and biofilm states and compared with three alternative chlorine-based biocides: neutral electrolyzed oxidizing water (NEOW), chlorine dioxide (CD) and sodium dichloroisocyanurate (NaDCC). The planktonic tests revealed that SH had the fastest antimicrobial action, NaDCC exhibited the highest antimicrobial rate and NEOW caused the highest antimicrobial effects. Additionally, NEOW was the biocide that allowed the highest formation of reactive oxygen species (ROS). In biofilm control, NEOW and CD were the most efficient biocides causing 3.26 and 3.20 log CFU·cm-2 reduction, respectively. In terms of stability for chlorine depletion, NEOW had the longest decay time for chlorine loss (70days at 5°C) and the lowest chlorine loss rate (0.013ppm·min-1 at 5°C). CD and NaDCC had equivalent stability. The overall results demonstrated NEOW as a good alternative to SH due to its higher antimicrobial effects and lower chlorine depletion over time.