Ilse Vandecandelaere

Marine aerobic biofilm as biocathode catalyst.

Stainless steel electrodes were immersed in open seawater and polarized for some days at -200 mV vs. Ag/AgCl. The current increase indicated the formation of biofilms that catalysed the electrochemical reduction of oxygen. These wild, electrochemically active (EA) biofilms were scraped, resuspended in seawater and used as the inoculum in closed 0.5L electrochemical reactors. This procedure allowed marine biofilms that are able to catalyse oxygen reduction to be formed in small, closed small vessels for the first time. Potential polarisation during biofilm formation was required to obtain EA biofilms and the roughness of the surface favoured high current values. The low availability of nutrients was shown to be a main limitation. Using an open reactor continuously fed with filtered seawater multiplied the current density by a factor of around 20, up to 60 microA/cm(2), which was higher than the current density provided in open seawater by the initial wild biofilm. These high values were attributed to continuous feeding with the nutrients contained in seawater and to suppression of the indigenous microbial species that compete with EA strains in natural open environments. Pure isolates were extracted from the wild biofilms and checked for EA properties. Of more than thirty different species tested, only Winogradskyella poriferorum and Acinetobacter johsonii gave current densities of respectively 7% and 3% of the current obtained with the wild biofilm used as inoculum. Current densities obtained with pure cultures were lower than those obtained with wild biofilms. It is suspected that synergic effects occur in whole biofilms or/and that wild strains may be more efficient than the cultured isolates.

Assessment of Microbial Diversity in Biofilms Recovered from Endotracheal Tubes Using Culture Dependent and Independent Approaches

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.

Electrochemical activity and bacterial diversity of natural marine biofilm in laboratory closed-systems.

Even if a widely shared mechanism actually does not exist, it is now generally accepted that, in aerobic conditions, marine electrochemically active biofilms (MEABs) induce faster oxygen reduction on stainless steel immersed in seawater. This phenomenon has been widely studied, but nearly all the experiments found in literature have been conducted in open-systems (i.e. experimental environments where seawater is constantly renewed). In this work we tried to obtain, in open circuit and potentiostatic conditions, MEABs in different laboratory closed-systems without water renewal (mesocosms), in order to verify the relationship between electrochemical activity and biofilm composition. The diversity of the microbial populations of biofilms obtained by our new kind of approach was examined by the DGGE technique (denaturing gradient gel electrophoresis). MEABs were obtained in all the mesocosms from 2000 to 2 L, showing in some cases electrochemical performances comparable to those of open-systems, and a very high genetic variability. Our DGGE results underline the difficulty in finding a correlation between electrochemical activity and composition of microbial populations.

Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater

Biofilms formed in aerobic seawater on stainless steel are known to be efficient catalysts of the electrochemical reduction of oxygen. Based on their genomic analysis, seven bacterial isolates were selected and a cyclic voltammetry (CV) procedure was implemented to check their electrocatalytic activity towards oxygen reduction. All isolates exhibited close catalytic characteristics. Comparison between CVs recorded with glassy carbon and pyrolytic graphite electrodes showed that the catalytic effect was not correlated with the surface area covered by the cells. The low catalytic effect obtained with filtered isolates indicated the involvement of released redox compounds, which was confirmed by CVs performed with adsorbed iron-porphyrin. None of the isolates were able to form electro-active biofilms under constant polarization. The capacity to catalyze oxygen reduction is shown to be a widespread property among bacteria, but the property detected by CV does not necessarily confer the ability to achieve stable oxygen reduction under constant polarization.

Leisingera aquimarina sp. nov., isolated from a marine electroactive biofilm, and emended descriptions of Leisingera methylohalidivorans Schaefer et al. 2002, Phaeobacter daeponensis Yoon et al. 2007 and Phaeobacter inhibens Martens et al. 2006

Strain LMG 24366(T) was isolated from a marine electroactive biofilm grown on a stainless steel cathode (Genova, Italy) and was investigated by using a polyphasic taxonomic approach. This study demonstrated that strain LMG 24366(T) represents a novel species within the genus Leisingera, which shared 98.9 % 16S rRNA gene similarity with its nearest phylogenetic neighbour, Leisingera methylohalidivorans. Strain LMG 24366(T) grew on betaine (1 mM) as a sole carbon source, whereas no growth was observed on L-methionine (10 mM). The phenotypic and genotypic analyses showed that strain LMG 24366(T) could be differentiated from established Leisingera species and that it represented a novel species, for which the name Leisingera aquimarina sp. nov. is proposed. The type strain is LMG 24366(T) (=CCUG 55860(T)) and has a DNA G+C content of 61.4 mol%.

Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

Microbial composition and antibiotic resistance of biofilms recovered from endotracheal tubes of mechanically ventilated patients

In critically ill patients, breathing is impaired and mechanical ventilation, using an endotracheal tube (ET) connected to a ventilator, is necessary. Although mechanical ventilation is a life-saving procedure, it is not without risk. Because of several reasons, a biofilm often forms at the distal end of the ET and this biofilm is a persistent source of bacteria which can infect the lungs, causing ventilator-associated pneumonia (VAP). There is a link between the microbial flora of ET biofilms and the microorganisms involved in the onset of VAP. Culture dependent and independent techniques were already used to identify the microbial flora of ET biofilms and also, the antibiotic resistance of microorganisms obtained from ET biofilms was determined. The ESKAPE pathogens play a dominant role in the onset of VAP and these organisms were frequently identified in ET biofilms. Also, antibiotic resistant microorganisms were frequently present in ET biofilms. Members of the normal oral flora were also identified in ET biofilms but it is thought that these organisms initiate ET biofilm formation and are not directly involved in the development of VAP.

The presence of antibiotic-resistant nosocomial pathogens in endotracheal tube biofilms and corresponding surveillance cultures

Mechanically ventilated patients often develop ventilator-associated pneumonia (VAP). Soon after intubation, a mixed biofilm harboring microbial pathogens is formed on the endotracheal tube (ET). It is believed that this biofilm contributes to the development of VAP. Unfortunately, the causative agent is often not known at the time VAP is suspected, and early therapy often relies on the identification of surveillance cultures (SC). It is thus important to know whether these SC can predict the microbial flora in ET biofilms. In this study, we compare the presence of a number of antibiotic-resistant nosocomial bacteria (Enterobacter aerogenes, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and of Candida albicans in cultures from ET biofilms and SC (i.e. sputum samples, nose swabs, and throat swabs) of 20 mechanically ventilated patients. Our data indicate that there is a good correlation between the presence of (antibiotic-resistant) pathogens in ET biofilms and SC.

A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification

We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (resazurin assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations.

Taking the Silver Bullet Colloidal Silver Particles for the Topical Treatment of Biofilm-Related Infections

Biofilms are aggregates of bacteria residing in a self-assembled matrix, which protects these sessile cells against external stress, including antibiotic therapies. In light of emerging multidrug-resistant bacteria, alternative strategies to antibiotics are emerging. The present study evaluated the activity of colloidal silver nanoparticles (AgNPs) of different shapes against biofilms formed by Staphylococcus aureus (SA), methicillin-resistant SA (MRSA), and Pseudomonas aeruginosa (PA). Colloidal quasi-spherical, cubic, and star-shaped AgNPs were synthesized, and their cytotoxicity on macrophages (THP-1) and bronchial epithelial cells (Nuli-1) was analyzed by the lactate dehydrogenase assay. The antibiofilm activity was assessed in vitro by the resazurin assay and in an in vivo infection model in Caenorhabditis elegans. Cubic and star-shaped AgNPs induced cytotoxicity, while quasi-spherical AgNPs were not toxic. Quasi-spherical AgNPs showed substantial antibiofilm activity in vitro with 96% (±2%), 97% (±1%), and 98% (±1%) biofilm killing of SA, MRSA, and PA, respectively, while significantly reducing mortality of infected nematodes. The in vivo antibiofilm activity was linked to the accumulation of AgNPs in the intestinal tract of C. elegans as observed by 3D X-ray tomography. Quasi-spherical AgNPs were physically stable in suspension for over 6 months with no observed loss in antibiofilm activity. While toxicity and stability limited the utilization of cubic and star-shaped AgNPs, quasi-spherical AgNPs could be rapidly synthesized, were stable and nontoxic, and showed substantial in vitro and in vivo activity against clinically relevant biofilms. Quasi-spherical AgNPs hold potential as pharmacotherapy, for example, as topical treatment for biofilm-related infections.