Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SpIF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, spIF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved

Draft genome sequence of methicillin-resistant Staphylococcus epidermidis strain ET-024, isolated from an endotracheal tube biofilm of a mechanically ventilated patient

Staphylococcus epidermidis strain ET-024 was isolated from a biofilm on an endotracheal tube of a mechanically ventilated patient. This strain is resistant to methicillin and the draft genome sequence shares some characteristics with other nosocomial S. epidermidis strains (such as S. epidermidis RP62A)

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 0.5 L 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 µA/cm2, 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 synergetic effects occur in whole biofilms or/and that wild strains may be more efficient than the cultured isolates

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

Biofilmsformed in aerobic seawater on stainless steel are known to be efficient catalysts of the electrochemicalreduction 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 oxygenreduction. 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-activebiofilms under constant polarization. The capacity to catalyze oxygenreduction is shown to be a widespread property among bacteria, but the property detected by CV does not necessarily confer the ability to achieve stable oxygenreduction under constant polarization

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

Bacterial diversity of a marine electroactive biofilm

Momenteel zijn fossiele brandstoffen de belangrijkste energiebronnen, maar de enorme uitstoot van koolstofdioxide is schadelijk en erg belastend voor het milieu. Daarnaast wordt er een uitputting van fossiele brandstoffen verwacht in de volgende decades. De zoektocht naar en de ontwikkeling van nieuwe en groene energiebronnen is van cruciaal belang om aan de alsmaar groeiende energievraag te voldoen. Eén van de mogelijkheden zijn microbiële brandstofcellen waarin bacteriën elektrische stroom produceren. Bacteriën katalyseren het elektronentransport naar of van de elektrode wat resulteert in stroomproductie. Deze bacteriën vormen hierbij elektroactieve biofilms (EAB) op het oppervlak van de elektrode. Voorgestelde studie kaderde in een Europees project getiteld “Electrochemical control of biofilm-forming micro-organisms: screening, identification and design of new knowledge-based technologies (EA-BIOFILM-508866 (NEST))”. Roestvrij staal coupons werden kathodisch gepolariseerd (-200 mV Ag/AgCl) in zeewater in de haven van Genua (Italië), wat resulteerde in stroomproductie (0.5 A/m²). De vorming van een biofilm werd waargenomen op het oppervlak van de kathode. De microbiële gemeenschap in deze mariene EAB werd onderzocht aan de hand van een reeks cultuurafhankelijke en cultuuronafhankelijke technieken, steunend op een polyfasische taxonomische aanpak. Driehonderd zesenvijftig isolaten werden opgepikt van drie verschillende groeimedia en werden allemaal geanalyseerd door middel van cellulaire vetzuuranalyse (FAME). De isolaten werden gegroepeerd op basis van hun FAME profielen en zo werden er twintig groepen, met ten minste 70 % profiel gelijkenis, afgebakend. De isolaten bleken te behoren tot verschillende fylogenetische groepen namelijk vertegenwoordigers van de klasse Alpha- (de genera Ruegeria, Phaeobacter en Leisingera) en Gammaproteobacteria (de genera Alteromonas, Pseudoalteromonas, Acinetobacter, Marinobacter, Idiomarina en Halomonas), het fylum Actinobacteria (de genera Arthrobacter en Frigoribacterium), de familie Flavobacteriaceae (de genera Maribacter and Winogradskyella) en het fylum Firmicutes (de genera Bacillus, Staphylococcus en Exiguobacterium) waren aanwezig. Deze hoge bacteriële diversiteit in de mariene EAB is in tegenspraak met eerdere studies van de microbiële gemeenschappen van EABs welke de aanrijking van specifieke bacteriële groepen beschreven. Deze tegenstelling kan verklaard worden door de korte incubatietijd (14 dagen) van de EAB van voorgestelde studie terwijl bij vorige studies de incubatietijd van de EABs varieerde van enkele maanden tot 5 jaar. De isolaten van twee FAME groepen (I en IV) waren dominant (45 % van alle isolaten) en werden in detail onderzocht aan de hand van BOX-PCR fingerprinting, DNA-DNA hybridisatie experimenten en fenotypische testen. Dit leidde tot de beschrijving van verschillende nieuwe bacteriële soorten. Eén nieuwe bacteriële soort kon beschreven worden binnen een bestaand genus van de klasse Gammaproteobacteria (FAME cluster I) namelijk Alteromonas genovensis sp. nov.. Gedetailleerd onderzoek van de isolaten van FAME cluster IV resulteerde in de beschrijving van 3 nieuwe bacteriële soorten binnen bestaande genera van de Roseobacter-groep van de klasse Alphaproteobacteria namelijk Ruegeria scottomollicae sp. nov., Phaeobacter caerulea sp. nov., Leisingera aquimarina sp. nov. en in de beschrijving van een nieuwe genus in de Roseobacter-groep namelijk Nautella italica gen. nov., sp. nov. Algemeen, de microbiële gemeenschap van de onderzochte mariene EAB was fylogenetisch erg divers en bestond uit typische mariene bacteriën. Grotendeels dezelfde fylogenetische groepen van bacteriën werden geïdentificeerd met cultuurafhankelijke en – onafhankelijke technieken, wat aantoont dat we erin geslaagd zijn om de dominante bacteriële gemeenschap op te kweken. Dus het toepassen van een reeks methodes met een verschillende taxonomische resolutie bleek erg nuttig te zijn om de diversiteit van deze mariene EAB gemeenschap te bestuderen

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