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Nosocomial infections have become a serious threat in our times and are getting more difficult to handle due to increasing development of resistances in bacteria. In this light, cold atmospheric plasma (CAP), which is known to effectively inactivate microorganisms, may be a promising alternative for application in the fields of dentistry and dermatology. CAPs are partly ionised gases, which operate at low temperature and are composed of electrons, ions, excited atoms and molecules, reactive oxygen and nitrogen species. In this study, the effect of CAP generated from ambient air was investigated against Enterococcus faecalis, grown on agar plates or as biofilms cultured for up to 72 h. CAP reduced the colony forming units (CFU) on agar plates by > 7 log10 steps. Treatment of 24 h old biofilms of E. faecalis resulted in CFU-reductions by ≥ 3 log10 steps after CAP treatment for 5 min and by ≥ 5 log10 steps after CAP treatment for 10 min. In biofilm experiments, chlorhexidine (CHX) and UVC radiation served as positive controls and were only slightly more effective than CAP. There was no damage of cytoplasmic membranes upon CAP treatment as shown by spectrometric measurements for release of nucleic acids. Thus, membrane damage seems not to be the primary mechanism of action for CAP towards E. faecalis. Overall, CAP showed pronounced antimicrobial efficacy against E. faecalis on agar plates as well as in biofilms similar to positive controls CHX or UVC., Competing Interests: The study employed a plasma source which had been developed and patented by terraplasma GmbH. The prime interest of terraplasma was to determine the safety of the plasma source in an independent study, carried out without undue influence by the company so that it provides a valuable documentation. The scientific value of this work lies in the possibility for advancing plasma treatment and providing the quantitative background for this future application, as well as paving the way for a clinical study. In this sense the interest is complementary – there is no competing interest. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

The importance of planktonic microbial communities is well acknowledged, since they are fundamental for several natural processes of aquatic ecosystems. Microorganisms naturally control the flux of nutrients, and also degrade and recycle anthropogenic organic and inorganic contaminants. Nevertheless, climate change effects and/or the runoff of nutrients/pollutants can affect the equilibrium of natural microbial communities influencing the occurrence of microbial pathogens and/or microbial toxin producers, which can compromise ecosystem environmental status. Therefore, improved microbial plankton monitoring is essential to better understand how these communities respond to environmental shifts. The study of marine microbial communities typically involves highly cost and time-consuming sampling procedures, which can limit the frequency of sampling and data availability. In this context, we developed and validated an in situ autonomous biosampler (IS-ABS) able to collect/concentrate in situ planktonic communities of different size fractions (targeting prokaryotes and unicellular eukaryotes) for posterior genomic, metagenomic, and/or transcriptomic analysis at a home laboratory. The IS-ABS field prototype is a small size and compact system able to operate up to 150 m depth. Water is pumped by a micropump (TCS MG2000) through a hydraulic circuit that allows in situ filtration of environmental water in one or more Sterivex filters placed in a filter cartridge. The IS-ABS also includes an application to program sampling definitions, allowing pre-setting configuration of the sampling. The efficiency of the IS-ABS was tested against traditional laboratory filtration standardized protocols. Results showed a good performance in terms of DNA recovery, as well as prokaryotic (16S rDNA) and eukaryotic (18S rDNA) community diversity analysis, using either methodologies. The IS-ABS automates the process of collecting environmental DNA, and is suitable for integration in water observation systems, what will contribute to substantially increase biological surveillances. Also, the use of highly sensitive genomic approaches allows a further study of the diversity and functions of whole or specific microbial communities., Competing Interests: The authors have declared that no competing interests exist.

There is a growing consensus that future technological developments of aquaculture systems should account for the structure and function of microbial communities in the whole system and not only in fish guts. In this study, we aimed to investigate the composition of bacterioplankton communities of a hatchery recirculating aquaculture system (RAS) used for the production of Senegalese sole (Solea senegalensis) juveniles. To this end, we used a 16S rRNA gene based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analyses to characterize the bacterioplankton communities of the RAS and its water supply. Overall, the most abundant orders were Alteromonadales, Rhodobacterales, Oceanospirillales, Vibrionales, Flavobacteriales, Lactobacillales, Thiotrichales, Burkholderiales and Bdellovibrionales. Although we found a clear distinction between the RAS and the water supply bacterioplankton communities, most of the abundant OTUs (≥50 sequences) in the hatchery RAS were also present in the water supply. These included OTUs related to Pseudoalteromonas genus and the Roseobacter clade, which are known to comprise bacterial members with activity against Vibrio fish pathogens. Overall, in contrast to previous findings for sole grow-out RAS, our results suggest that the water supply may influence the bacterioplankton community structure of sole hatchery RAS. Further studies are needed to investigate the effect of aquaculture practices on RAS bacterioplankton communities and identification of the key drivers of their structure and diversity., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP) and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST) combined with the AMP temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and tachyplesin I linear analogue (TP-I-L) was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated.

Mesoscale circulation generated by the Loop Current in the Northern Gulf of Mexico (NGOM) delivers growth-limiting nutrients to the microbial plankton of the euphotic zone. Consequences of physicochemically driven community shifts on higher order consumers and subsequent impacts on the biological carbon pump remain poorly understood. This study evaluates microbial plankton <10 μm abundance and community structure across both cyclonic and anti-cyclonic circulation features in the NGOM using flow cytometry (SYBR Green I and autofluorescence parameters). Non-parametric multivariate hierarchical cluster analyses indicated that significant spatial variability in community structure exists such that stations that clustered together were defined as having a specific 'microbial signature' (i.e. statistically homogeneous community structure profiles based on relative abundance of microbial groups). Salinity and a combination of sea surface height anomaly and sea surface temperature were determined by distance based linear modeling to be abiotic predictor variables significantly correlated to changes in microbial signatures. Correlations between increased microbial abundance and availability of nitrogen suggest nitrogen-limitation of microbial plankton in this open ocean area. Regions of combined coastal water entrainment and mesoscale convergence corresponded to increased heterotrophic prokaryote abundance relative to autotrophic plankton. The results provide an initial assessment of how mesoscale circulation potentially influences microbial plankton abundance and community structure in the NGOM.

Carbon steels are widely used in the oil and gas industry from downhole tubing to transport trunk lines. Microbes form biofilms, some of which cause the so-called microbiologically influenced corrosion (MIC) of carbon steels. MIC by sulfate reducing bacteria (SRB) is often a leading cause in MIC failures. Electrogenic SRB sessile cells harvest extracellular electrons from elemental iron oxidation for energy production in their metabolism. A previous study suggested that electron mediators riboflavin and flavin adenine dinucleotide (FAD) both accelerated the MIC of 304 stainless steel by the Desulfovibrio vulgaris biofilm that is a corrosive SRB biofilm. Compared with stainless steels, carbon steels are usually far more prone to SRB attacks because SRB biofilms form much denser biofilms on carbon steel surfaces with a sessile cell density that is two orders of magnitude higher. In this work, C1018 carbon steel coupons were used in tests of MIC by D. vulgaris with and without an electron mediator. Experimental weight loss and pit depth data conclusively confirmed that both riboflavin and FAD were able to accelerate D. vulgaris attack against the carbon steel considerably. It has important implications in MIC failure analysis and MIC mitigation in the oil and gas industry.

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

The submarine volcanic eruption occurring near El Hierro (Canary Islands) in October 2011 provided a unique opportunity to determine the effects of such events on the microbial populations of the surrounding waters. The birth of a new underwater volcano produced a large plume of vent material detectable from space that led to abrupt changes in the physical-chemical properties of the water column. We combined flow cytometry and 454-pyrosequencing of 16S rRNA gene amplicons (V1-V3 regions for Bacteria and V3-V5 for Archaea) to monitor the area around the volcano through the eruptive and post-eruptive phases (November 2011 to April 2012). Flow cytometric analyses revealed higher abundance and relative activity (expressed as a percentage of high-nucleic acid content cells) of heterotrophic prokaryotes during the eruptive process as compared to post-eruptive stages. Changes observed in populations detectable by flow cytometry were more evident at depths closer to the volcano (~70-200 m), coinciding also with oxygen depletion. Alpha-diversity analyses revealed that species richness (Chao1 index) decreased during the eruptive phase; however, no dramatic changes in community composition were observed. The most abundant taxa during the eruptive phase were similar to those in the post-eruptive stages and to those typically prevalent in oceanic bacterioplankton communities (i.e. the alphaproteobacterial SAR11 group, the Flavobacteriia class of the Bacteroidetes and certain groups of Gammaproteobacteria). Yet, although at low abundance, we also detected the presence of taxa not typically found in bacterioplankton communities such as the Epsilonproteobacteria and members of the candidate division ZB3, particularly during the eruptive stage. These groups are often associated with deep-sea hydrothermal vents or sulfur-rich springs. Both cytometric and sequence analyses showed that once the eruption ceased, evidences of the volcano-induced changes were no longer observed.

Biofilm formation by food-related bacteria and food-related pathogenesis are significant problems in the food industry. Even though much disinfection and mechanical procedure exist for removal of biofilms, they may fail to eliminate pre-established biofilms. cis-2 decenoic acid (CDA), an unsaturated fatty acid messenger produced by Pseudomonas aeruginosa, is reportedly capable of inducing the dispersion of established biofilms by multiple types of microorganisms. However, whether CDA has potential to boost the actions of certain antimicrobials is unknown. Here, the activity of CDA as an inducer of pre-established biofilms dispersal, formed by four main food pathogens; Staphylococcus aureus, Bacillus cereus, Salmonella enterica and E. coli, was measured using both semi-batch and continuous cultures bioassays. To assess the ability of CDA combined biocides treatments to remove pre-established biofilms formed on stainless steel discs, CFU counts were performed for both treated and untreated cultures. Eradication of the biofilms by CDA combined antibiotics was evaluated using crystal violet staining. The effect of CDA combined treatments (antibiotics and disinfectants) on biofilm surface area and bacteria viability was evaluated using fluorescence microscopy, digital image analysis and LIVE/DEAD staining. MICs were also determined to assess the probable inhibitory effects of CDA combined treatments on the growth of tested microorganisms' planktonic cells. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least two-fold increase in the number of planktonic cells in all cultures. While antibiotics or disinfectants alone exerted a trivial effect on CFU counts and percentage of surface area covered by the biofilms, combinational treatments with both 310 nM CDA and antibiotics or disinfectants led to approximate 80% reduction in biofilm biomass. These data suggests that combined treatments with CDA would pave the way toward developing new strategies to control biofilms with widespread applications in industry as well as medicine.

This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ.

Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.

There is increasing recognition that stochasticity involved in gene regulatory processes may help cells enhance the signal or synchronize expression for a group of genes. Thus the validity of the traditional deterministic approach to modeling the foregoing processes cannot be without exception. In this study, we identify a frequently encountered situation, i.e., the biofilm, which has in the past been persistently investigated with intracellular deterministic models in the literature. We show in this paper circumstances in which use of the intracellular deterministic model appears distinctly inappropriate. In Enterococcus faecalis, the horizontal gene transfer of plasmid spreads drug resistance. The induction of conjugation in planktonic and biofilm circumstances is examined here with stochastic as well as deterministic models. The stochastic model is formulated with the Chemical Master Equation (CME) for planktonic cells and Reaction-Diffusion Master Equation (RDME) for biofilm. The results show that although the deterministic model works well for the perfectly-mixed planktonic circumstance, it fails to predict the averaged behavior in the biofilm, a behavior that has come to be known as stochastic focusing. A notable finding from this work is that the interception of antagonistic feedback loops to signaling, accentuates stochastic focusing. Moreover, interestingly, increasing particle number of a control variable could lead to an even larger deviation. Intracellular stochasticity plays an important role in biofilm and we surmise by implications from the model, that cell populations may use it to minimize the influence from environmental fluctuation.

In order to characterize how disturbances to microbial communities are propagated over temporal and spatial scales in aquatic environments, the dynamics of bacterial assemblages throughout a subtropical coastal embayment were investigated via SSU rRNA gene analyses over an 8-month period, which encompassed a large storm event. During non-perturbed conditions, sampling sites clustered into three groups based on their microbial community composition: an offshore oceanic group, a freshwater group, and a distinct and persistent coastal group. Significant differences in measured environmental parameters or in the bacterial community due to the storm event were found only within the coastal cluster of sampling sites, and only at 5 of 12 locations; three of these sites showed a significant response in both environmental and bacterial community characteristics. These responses were most pronounced at sites close to the shoreline. During the storm event, otherwise common bacterioplankton community members such as marine Synechococcus sp. and members of the SAR11 clade of Alphaproteobacteria decreased in relative abundance in the affected coastal zone, whereas several lineages of Gammaproteobacteria, Betaproteobacteria, and members of the Roseobacter clade of Alphaproteobacteria increased. The complex spatial patterns in both environmental conditions and microbial community structure related to freshwater runoff and wind convection during the perturbation event leads us to conclude that spatial heterogeneity was an important factor influencing both the dynamics and the resistance of the bacterioplankton communities to disturbances throughout this complex subtropical coastal system. This heterogeneity may play a role in facilitating a rapid rebound of regions harboring distinctly coastal bacterioplankton communities to their pre-disturbed taxonomic composition.

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

In the Gulf of Maine area (GoMA), as elsewhere in the ocean, the organisms of greatest numerical abundance are microbes. Viruses in GoMA are largely cyanophages and bacteriophages, including podoviruses which lack tails. There is also evidence of Mimivirus and Chlorovirus in the metagenome. Bacteria in GoMA comprise the dominant SAR11 phylotype cluster, and other abundant phylotypes such as SAR86-like cluster, SAR116-like cluster, Roseobacter, Rhodospirillaceae, Acidomicrobidae, Flavobacteriales, Cytophaga, and unclassified Alphaproteobacteria and Gammaproteobacteria clusters. Bacterial epibionts of the dinoflagellate Alexandrium fundyense include Rhodobacteraceae, Flavobacteriaceae, Cytophaga spp., Sulfitobacter spp., Sphingomonas spp., and unclassified Bacteroidetes. Phototrophic prokaryotes in GoMA include cyanobacteria that contain chlorophyll (mainly Synechococcus), aerobic anoxygenic phototrophs that contain bacteriochlorophyll, and bacteria that contain proteorhodopsin. Eukaryotic microalgae in GoMA include Bacillariophyceae, Dinophyceae, Prymnesiophyceae, Prasinophyceae, Trebouxiophyceae, Cryptophyceae, Dictyochophyceae, Chrysophyceae, Eustigmatophyceae, Pelagophyceae, Synurophyceae, and Xanthophyceae. There are no records of Bolidophyceae, Aurearenophyceae, Raphidophyceae, and Synchromophyceae in GoMA. In total, there are records for 665 names and 229 genera of microalgae. Heterotrophic eukaryotic protists in GoMA include Dinophyceae, Alveolata, Apicomplexa, amoeboid organisms, Labrynthulida, and heterotrophic marine stramenopiles (MAST). Ciliates include Strombidium, Lohmaniella, Tontonia, Strobilidium, Strombidinopsis and the mixotrophs Laboea strobila and Myrionecta rubrum (ex Mesodinium rubra). An inventory of selected microbial groups in each of 14 physiographic regions in GoMA is made by combining information on the depth-dependent variation of cell density and the depth-dependent variation of water volume. Across the entire GoMA, an estimate for the minimum abundance of cell-based microbes is 1.7×10(25) organisms. By one account, this number of microbes implies a richness of 10(5) to 10(6) taxa in the entire water volume of GoMA. Morphological diversity in microplankton is well-described but the true extent of taxonomic diversity, especially in the femtoplankton, picoplankton and nanoplankton--whether autotrophic, heterotrophic, or mixotrophic, is unknown.

Eukaryotic microorganisms have been undersampled in biodiversity studies in freshwater environments. We present an original 18S rDNA survey of freshwater picoeukaryotes sampled during spring/summer 2005, complementing an earlier study conducted in autumn 2004 in Lake Pavin (France). These studies were designed to detect the small unidentified heterotrophic flagellates (HF, 0.6-5 microm) which are considered the main bacterivores in aquatic systems. Alveolates, Fungi and Stramenopiles represented 65% of the total diversity and differed from the dominant groups known from microscopic studies. Fungi and Telonemia taxa were restricted to the oxic zone which displayed two fold more operational taxonomic units (OTUs) than the oxycline. Temporal forcing also appeared as a driving force in the diversification within targeted organisms. Several sequences were not similar to those in databases and were considered as new or unsampled taxa, some of which may be typical of freshwater environments. Two taxa known from marine systems, the genera Telonema and Amoebophrya, were retrieved for the first time in our freshwater study. The analysis of potential trophic strategies displayed among the targeted HF highlighted the dominance of parasites and saprotrophs, and provided indications that these organisms have probably been wrongfully regarded as bacterivores in previous studies. A theoretical exercise based on a new 'parasite/saprotroph-dominated HF hypothesis' demonstrates that the inclusion of parasites and saprotrophs may increase the functional role of the microbial loop as a link for carbon flows in pelagic ecosystems. New interesting perspectives in aquatic microbial ecology are thus opened.