High resolution ITS amplicon melting analysis as a tool to analyse microbial communities of household biofilms in ex-situ models.

Biofilms are the most common growth types of microorganisms. These complex communities usually consist of different species and are embedded in an extracellular matrix containing polymers, proteins and DNA. This matrix offers protection against different (a)biotic environmental factors and generally increases resistances. Higher resistances against antibiotics are one of the main reasons why biofilms are often associated with healthcare settings. Nevertheless, they are also found in domestic settings, mostly in humid places with abundant nutrients like dishwashers or washing machines. Biofilms in these areas show individual compositions and are influenced for example by temperature, frequency of use or the age of the device. In this study, we introduce a model for the ex-situ cultivation of domestic biofilms from household appliances. Furthermore, we tested the ability of high resolution melting analysis (HRMA) as a tool for analysing these biofilms. Our goal was to maintain a high amount of complexity in the ex-situ biofilms that is characterized by the melting behavior of the contained DNA. Dishwasher and washing machine biofilms were sampled in private households and cultivated for 10 d. After DNA extraction, 16S rDNA was sequenced and melting behavior of the bacterial Internal Transcribed Spacer (ITS) region was analysed. Additionally, testing for independence of continuous new sampling, storage of cultivated biofilms in glycerol stocks and following recultivation of them was done up to three times. Our results show that a high level of complexity could be maintained in the ex-situ biofilms after 10 d of cultivation, although in general the bacterial diversity slightly decreased compared to the original biofilm in most cases. Recultivation of a similar biofilm from glycerol stocks was possible as well with some impact by various factors. Differences in the bacterial composition of biofilms could clearly made visible by HRMA although it was not possible to match peaks to a specific phylogenetic group. Still, HRMA proved to be a less costly and time consuming alternative to sequencing for the characterization of biofilms.

The biofilm matrix: multitasking in a shared space.

The biofilm matrix can be considered to be a shared space for the encased microbial cells, comprising a wide variety of extracellular polymeric substances (EPS), such as polysaccharides, proteins, amyloids, lipids and extracellular DNA (eDNA), as well as membrane vesicles and humic-like microbially derived refractory substances. EPS are dynamic in space and time and their components interact in complex ways, fulfilling various functions: to stabilize the matrix, acquire nutrients, retain and protect eDNA or exoenzymes, or offer sorption sites for ions and hydrophobic substances. The retention of exoenzymes effectively renders the biofilm matrix an external digestion system influencing the global turnover of biopolymers, considering the ubiquitous relevance of biofilms. Physico-chemical and biological interactions and environmental conditions enable biofilm systems to morph into films, microcolonies and macrocolonies, films, ridges, ripples, columns, pellicles, bubbles, mushrooms and suspended aggregates - in response to the very diverse conditions confronting a particular biofilm community. Assembly and dynamics of the matrix are mostly coordinated by secondary messengers, signalling molecules or small RNAs, in both medically relevant and environmental biofilms. Fully deciphering how bacteria provide structure to the matrix, and thus facilitate and benefit from extracellular reactions, remains the challenge for future biofilm research.

A Comprehensive View of Microbial Communities in the Laundering Cycle Suggests a Preventive Effect of Soil Bacteria on Malodour Formation.

Microorganisms are an important factor in the wash-and-use cycle of textiles since they can cause unwanted aesthetic effects, such as malodour formation, and even pose health risks. In this regard, a comprehensive view of the microbial communities in washing machines and consideration of the microbial contamination of used textiles is needed to understand the formation of malodour and evaluate the infection risk related to laundering. So far, neither the compositions of washing machine biofilms leading to the formation of or protection against malodour have been investigated intensively, nor have microbial communities on used towels been analysed after normal use. Our results link the qualitative and quantitative analysis of microbial communities in washing machines and on used towels with the occurrence of malodour and thus not only allow for a better risk evaluation but also suggest bacterial colonizers of washing machines that might prevent malodour formation. It was shown that soil bacteria such as Rhizobium, Agrobacterium, Bosea, and Microbacterium in particular are found in non-odourous machines, and that Rhizobium species are able to prevent malodour formation in an in vitro model.

Controlling the hydraulic resistance of membrane biofilms by engineering biofilm physical structure.

The application of membrane technology for water treatment and reuse is hampered by the development of a microbial biofilm. Biofilm growth in micro-and ultrafiltration (MF/UF) membrane modules, on both the membrane surface and feed spacer, can form a secondary membrane and exert resistance to permeation and crossflow, increasing energy demand and decreasing permeate quantity and quality. In recent years, exhaustive efforts were made to understand the chemical, structural and hydraulic characteristics of membrane biofilms. In this review, we critically assess which specific structural features of membrane biofilms exert resistance to forced water passage in MF/UF membranes systems applied to water and wastewater treatment, and how biofilm physical structure can be engineered by process operation to impose less hydraulic resistance ("below-the-pain threshold"). Counter-intuitively, biofilms with greater thickness do not always cause a higher hydraulic resistance than thinner biofilms. Dense biofilms, however, had consistently higher hydraulic resistances compared to less dense biofilms. The mechanism by which density exerts hydraulic resistance is reported in the literature to be dependant on the biofilms' internal packing structure and EPS chemical composition (e.g., porosity, polymer concentration). Current reports of internal porosity in membrane biofilms are not supported by adequate experimental evidence or by a reliable methodology, limiting a unified understanding of biofilm internal structure. Identifying the dependency of hydraulic resistance on biofilm density invites efforts to control the hydraulic resistance of membrane biofilms by engineering internal biofilm structure. Regulation of biofilm internal structure is possible by alteration of key determinants such as feed water nutrient composition/concentration, hydraulic shear stress and resistance and can engineer biofilm structural development to decrease density and therein hydraulic resistance. Future efforts should seek to determine the extent to which the concept of "biofilm engineering" can be extended to other biofilm parameters such as mechanical stability and the implication for biofilm control/removal in engineered water systems (e.g., pipelines and/or, cooling towers) susceptible to biofouling.

Water in bacterial biofilms: pores and channels, storage and transport functions.

Bacterial biofilms occur in many natural and industrial environments. Besides bacteria, biofilms comprise over 70 wt% water. Water in biofilms occurs as bound- or free-water. Bound-water is adsorbed to bacterial surfaces or biofilm (matrix) structures and possesses different Infra-red and Nuclear-Magnetic-Resonance signatures than free-water. Bound-water is different from intra-cellularly confined-water or water confined within biofilm structures and bacteria are actively involved in building water-filled structures by bacterial swimmers, dispersion or lytic self-sacrifice. Water-filled structures can be transient due to blocking, resulting from bacterial growth, compression or additional matrix formation and are generally referred to as "channels and pores." Channels and pores can be distinguished based on mechanism of formation, function and dimension. Channels allow transport of nutrients, waste-products, signalling molecules and antibiotics through a biofilm provided the cargo does not adsorb to channel walls and channels have a large length/width ratio. Pores serve a storage function for nutrients and dilute waste-products or antimicrobials and thus should have a length/width ratio close to unity. The understanding provided here on the role of water in biofilms, can be employed to artificially engineer by-pass channels or additional pores in industrial and environmental biofilms to increase production yields or enhance antimicrobial penetration in infectious biofilms.

Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80°C and pH 2 to 3. Due to its unique physiological properties, allowing life at environmental extremes, and the recent availability of genetic tools, this extremophile has received increasing interest for biotechnological applications. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius toward the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred at up to 1.5% (vol/vol) 1-butanol, while planktonic growth was observed at up to 1% (vol/vol) 1-butanol. Confocal laser-scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy, three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in the absence and presence of 1-butanol. In response to 1% (vol/vol) 1-butanol, transcript levels of genes encoding motility and cell envelope structures, as well as membrane proteins, were reduced. Cell division and/or vesicle formation were upregulated. Furthermore, changes in immune and defense systems, as well as metabolism and general stress responses, were observed. Our findings show that the extreme lifestyle of S.acidocaldarius coincided with a high tolerance to organic solvents. This study provides what may be the first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldariusIMPORTANCEArchaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius, which is adapted to the two extremes of low pH and high temperature, to study its tolerance and robustness as well as its global cellular response toward organic solvents, as exemplified by 1-butanol. We were able to identify biofilm formation as a primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes, such as cell division and cellular defense systems, thus suggesting a global response for the protection at the population level.

Who put the film in biofilm? The migration of a term from wastewater engineering to medicine and beyond.

Sessile microorganisms were described as early as the seventeenth century. However, the term biofilm arose only in the 1960s in wastewater treatment research and was adopted later in marine fouling and in medical and dental microbiology. The sessile mode of microbial life was gradually recognized to be predominant on Earth, and the term biofilm became established for the growth of microorganisms in aggregates, frequently associated with interfaces, although many, if not the majority, of them not being continuous "films" in the strict sense. In this sessile form of life, microorganisms live in close proximity in a matrix of extracellular polymeric substances (EPS). They share emerging properties, clearly distinct from solitary free floating planktonic microbial cells. Common characteristics include the formation of synergistic microconsortia, using the EPS matrix as an external digestion system, the formation of gradients and high biodiversity over microscopically small distances, resource capture and retention, facilitated gene exchange as well as intercellular communication, and enhanced tolerance to antimicrobials. Thus, biofilms belong to the class of collective systems in biology, like forests, beehives, or coral reefs, although the term film addresses only one form of the various manifestations of microbial aggregates. The uncertainty of this term is discussed, and it is acknowledged that it will not likely be replaced soon, but it is recommended to understand these communities in the broader sense of microbial aggregates.

Nitrifying niche differentiation in biofilms from full-scale chloraminated drinking water distribution system.

Tropical conditions favour the auto-decomposition of monochloramine (MCA) leading to disinfectant decay and free ammonia in drinking water distribution systems (DWDS); thus, they promote the growth of nitrifiers and the development of biofilms on the inner-pipe surface. Biofilms can adversely impact the provision of safe and biologically stable water. Moreover, there is a general lack of understanding of the role of microbial communities in DWDS in regions with warm temperatures and no distinct seasons. Here, we report a survey on biofilms from full-scale monochloraminated DWDS in a highly urbanised metropolis using next generation sequencing tools. The monitoring campaign consisted of sampling biofilms and bulk waters from 21 in-service pipes. We characterized the microbial community with emphasis on nitrifying bacteria and archaea using 16S rRNA gene amplicon sequencing and potential nitrification activity. Samples grouped into two clusters, characterized by their low (Cluster LD) and high (Cluster HD) α-diversity. Both clusters harbour microorganisms related to nitrification: i) Nitrosomonas (24.9-68.8%), an ammonia oxidising bacterium (AOB) that dominated Cluster LD, and ii) a co-aggregation of genus Nitrospira (9.8-32.5%), a nitrite oxidising bacterium (NOB), and Thaumarchaeota (1.4-10.9%), chemolithotrophic ammonia oxidising (AOA) archaea that were among the most abundant OTUs in Cluster HD. Activity tests performed with fresh biofilm samples confirmed that these two clusters represent distinctive biofilm niches performing different stages of the nitrification process. Cluster LD correlated with a high concentration of MCA, which caused dysbiosis and resulted in high unevenness of the cluster. In cluster HD, with more biomass, chemical reactions involving nitrite increased the MCA demand, releasing ammonia and allowing more nitrifiers to grow, like AOA and NOB. From this study, we conclude that an MCA residual gradient along the DWDS drives and shapes the microbial community assembly and should be considered when designing effective disinfection strategies.

Biofouling and me: My Stockholm syndrome with biofilms.

Biofouling is the undesired deposition and growth of microorganisms on surfaces, forming biofilms. The definition is subjective and operational: not every biofilm causes biofouling - only if a given a subjective "threshold of interference" is exceeded, biofilms cause technical or medical problems. These range from the formation of slime layers on ship hulls or in pipelines, which increase friction resistance, to separation membranes, on which biofilms increase hydraulic resistance, to heat exchangers where they interfere with heat transport to contamination of treated water by eroded biofilm cells which may comprise hygienically relevant microorganisms, and, most dangerous, to biofilms on implants and catheters which can cause persistent infections. The largest fraction of anti-fouling research, usually in short-term experiments, is focused on prevention or limiting primary microbial adhesion. Intuitively, this appears only logical, but turns out mostly hopeless. This is because in technical systems with open access for microorganisms, all surfaces are colonized sooner or later which explains the very limited success of that research. As a result, the use of biocides remains the major tool to fight persistent biofilms. However, this is costly in terms of biocides, it stresses working materials, causes off-time and environmental damage and it usually leaves large parts of biofilms in place, ready for regrowth. In order to really solve biofouling problems, it is necessary to learn how to live with biofilms and mitigate their detrimental effects. This requires rather an integrated strategy than aiming to invent "one-shot" solutions. In this context, it helps to understand the biofilm way of life as a natural phenomenon. Biofilms are the oldest, most successful and most widely distributed form of life on earth, existing even in extreme environments and being highly resilient. Microorganisms in biofilms live in a self-produced matrix of extracellular polymeric substances (EPS) which allows them to develop emerging properties such as enhanced nutrient acquisition, synergistic microconsortia, enhanced tolerance to biocides and antibiotics, intense intercellular communication and cooperation. Transiently immobilized, biofilm organisms turn their matrix into an external digestion system by retaining complexed exoenzymes in the matrix. Biofilms grow even on traces of any biodegradable material, therefore, an effective anti-fouling strategy comprises to keep the system low in nutrients (good housekeeping), employing low-fouling, easy-to-clean surfaces, monitoring of biofilm development, allowing for early intervention, and acknowledging that cleaning can be more important than trying to kill biofilms, because cleaning does not cut the nutrient supply of survivors and dead biomass serves as an additional carbon source for "cannibalizing" survivors, supporting rapid after growth. An integrated concept is presented as the result of a long journey of the author through biofouling problems.

Tolerances of Deinococcus geothermalis Biofilms and Planktonic Cells Exposed to Space and Simulated Martian Conditions in Low Earth Orbit for Almost Two Years.

Fossilized biofilms represent one of the oldest known confirmations of life on the Earth. The success of microbes in biofilms results from properties that are inherent in the biofilm, including enhanced interaction, protection, and biodiversity. Given the diversity of microbes that live in biofilms in harsh environments on the Earth, it is logical to hypothesize that, if microbes inhabit other bodies in the Universe, there are also biofilms on those bodies. The Biofilm Organisms Surfing Space experiment was conducted as part of the EXPOSE-R2 mission on the International Space Station. The experiment was an international collaboration designed to perform a comparative study regarding the survival of biofilms versus planktonic cells of various microorganisms, exposed to space and Mars-like conditions. The objective was to determine whether there are lifestyle-dependent differences to cope with the unique mixture of stress factors, including desiccation, temperature oscillations, vacuum, or a Mars-like gas atmosphere and pressure in combination with extraterrestrial or Mars-like ultraviolet (UV) radiation residing during the long-term space mission. In this study, the outcome of the flight and mission ground reference analysis of Deinococcus geothermalis is presented. Cultural tests demonstrated that D. geothermalis remained viable in the desiccated state, being able to survive space and Mars-like conditions and tolerating high extraterrestrial UV radiation for more than 2 years. Culturability decreased, but was better preserved, in the biofilm consortium than in planktonic cells. These results are correlated to differences in genomic integrity after exposure, as visualized by random amplified polymorphic DNA-polymerase chain reaction. Interestingly, cultivation-independent viability markers such as membrane integrity, ATP content, and intracellular esterase activity remained nearly unaffected, indicating that subpopulations of the cells had survived in a viable but nonculturable state. These findings support the hypothesis of long-term survival of microorganisms under the harsh environmental conditions in space and on Mars to a higher degree if exposed as biofilm.

Bacteria and archaea on Earth and their abundance in biofilms.

Biofilms are a form of collective life with emergent properties that confer many advantages on their inhabitants, and they represent a much higher level of organization than single cells do. However, to date, no global analysis on biofilm abundance exists. We offer a critical discussion of the definition of biofilms and compile current estimates of global cell numbers in major microbial habitats, mindful of the associated uncertainty. Most bacteria and archaea on Earth (1.2 × 1030 cells) exist in the 'big five' habitats: deep oceanic subsurface (4 × 1029), upper oceanic sediment (5 × 1028), deep continental subsurface (3 × 1029), soil (3 × 1029) and oceans (1 × 1029). The remaining habitats, including groundwater, the atmosphere, the ocean surface microlayer, humans, animals and the phyllosphere, account for fewer cells by orders of magnitude. Biofilms dominate in all habitats on the surface of the Earth, except in the oceans, accounting for ~80% of bacterial and archaeal cells. In the deep subsurface, however, they cannot always be distinguished from single sessile cells; we estimate that 20-80% of cells in the subsurface exist as biofilms. Hence, overall, 40-80% of cells on Earth reside in biofilms. We conclude that biofilms drive all biogeochemical processes and represent the main way of active bacterial and archaeal life.

Extracellular polymeric substances of biofilms: Suffering from an identity crisis.

Microbial biofilms can be both cause and cure to a range of emerging societal problems including antimicrobial tolerance, water sanitation, water scarcity and pollution. The identities of extracellular polymeric substances (EPS) responsible for the establishment and function of biofilms are poorly understood. The lack of information on the chemical and physical identities of EPS limits the potential to rationally engineer biofilm processes, and impedes progress within the water and wastewater sector towards a circular economy and resource recovery. Here, a multidisciplinary roadmap for addressing this EPS identity crisis is proposed. This involves improved EPS extraction and characterization methodologies, cross-referencing between model biofilms and full-scale biofilm systems, and functional description of isolated EPS with in situ techniques (e.g. microscopy) coupled with genomics, proteomics and glycomics. The current extraction and spectrophotometric characterization methods, often based on the principle not to compromise the integrity of the microbial cells, should be critically assessed, and more comprehensive methods for recovery and characterization of EPS need to be developed.

Survival of Deinococcus geothermalis in Biofilms under Desiccation and Simulated Space and Martian Conditions.

Biofilm formation represents a successful survival strategy for bacteria. In biofilms, cells are embedded in a matrix of extracellular polymeric substances (EPS). As they are often more stress-tolerant than single cells, biofilm cells might survive the conditions present in space and on Mars. To investigate this topic, the bacterium Deinococcus geothermalis was chosen as a model organism due to its tolerance toward desiccation and radiation. Biofilms cultivated on membranes and, for comparison, planktonically grown cells deposited on membranes were air-dried and exposed to individual stressors that included prolonged desiccation, extreme temperatures, vacuum, simulated martian atmosphere, and UV irradiation, and they were exposed to combinations of stressors that simulate space (desiccation + vacuum + UV) or martian (desiccation + Mars atmosphere + UV) conditions. The effect of sulfatic Mars regolith simulant on cell viability during stress was investigated separately. The EPS produced by the biofilm cells contained mainly polysaccharides and proteins. To detect viable but nonculturable (VBNC) cells, cultivation-independent viability indicators (membrane integrity, ATP, 16S rRNA) were determined in addition to colony counts. Desiccation for 2 months resulted in a decrease of culturability with minor changes of membrane integrity in biofilm cells and major loss of membrane integrity in planktonic bacteria. Temperatures between -25°C and +60°C, vacuum, and Mars atmosphere affected neither culturability nor membrane integrity in both phenotypes. Monochromatic (254 nm; ≥1 kJ m-2) and polychromatic (200-400 nm; >5.5 MJ m-2 for planktonic cells and >270 MJ m-2 for biofilms) UV irradiation significantly reduced the culturability of D. geothermalis but did not affect cultivation-independent viability markers, indicating the induction of a VBNC state in UV-irradiated cells. In conclusion, a substantial proportion of the D. geothermalis population remained viable under all stress conditions tested, and in most cases the biofilm form proved advantageous for surviving space and Mars-like conditions. Key Words: Biofilms-Desiccation-UV radiation-Mars-Lithopanspermia. Astrobiology 17, 431-447.

EPS-Then and Now.

"Slime" played a brief and spectacular role in the 19th century founded by the theory of primordial slime by Ernst Haeckel. However, that substance was never found and eventually abandoned. Further scientific attention slowly began in the 1930s referring to slime as a microbial product and then was inspired by "How bacteria stick" by Costerton et al. in 1978, and the matrix material was considered to be polysaccharides. Later, it turned out that proteins, nucleic acids and lipids were major other constituents of the extracellular polymeric substances (EPS), an acronym which was highly discussed. The role of the EPS matrix turns out to be fundamental for biofilms, in terms of keeping cells in proximity and allowing for extended interaction, resource capture, mechanical strength and other properties, which emerge from the life of biofilm organisms, including enhanced tolerance to antimicrobials and other stress. The EPS components are extremely complex and dynamic and fulfil many functional roles, turning biofilms into the most ubiquitous and successful form of life on Earth.

Post-industrial river water quality-Fit for bathing again?

For the Ruhr River, bathing has been prohibited for decades. However, along with significant improvements of the hygienic water quality, there is an increasing demand of using the river for recreational purposes, in particular for bathing. In the "Safe Ruhr" interdisciplinary research project, demands, options and chances for lifting the bathing ban for the Ruhr River were investigated. As being the prominent reason for persisting recreational restrictions, microbiological water quality was in the focus of interest. Not only the faecal indicator organisms (FIOs) as required by the European Bathing Water Directive were considered, but also pathogens such as Salmonella, Pseudomonas aeruginosa, Legionella pneumophila, Campylobacter, Leptospira, enteroviruses and protozoan parasites. In this introductory paper, we firstly relate current recreational desires to historical experiences of river bathing. After recapitulating relevant microbial river contamination sources (predominantly sewage treatment plants, combined sewer overflows, and surface runoffs), we review existing knowledge about the relationships of FIOs and pathogens in rivers designated for recreational purposes, and then trace the evolution, rationale and validity of recreational freshwater quality criteria which are, despite obvious uncertainties, mostly relying on the FIO paradigm. In particular, the representativeness of FIOs is critically discussed. The working programme of Safe Ruhr, aiming at initiating and facilitating a process towards legalisation of Ruhr River bathing, is outlined. Sources of contamination can be technically handled which leaves the actual measures to political decisions. As contaminations are transient, only occasionally exceeding legal limits, a flexible bathing site management, warning bathers of non-safe situations, may amend technical interventions and offer innovative solutions. As a result, a situation-adapted system for lifting of the bathing ban for Ruhr River appears realistic.

Biofilms: an emergent form of bacterial life.

Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecological perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecological success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.

Nanosilver induces a non-culturable but metabolically active state in Pseudomonas aeruginosa.

The antimicrobial properties of silver nanoparticles (AgNPs) have raised expectations for the protection of medical devices and consumer products against biofilms. The effect of silver on bacteria is commonly determined by culture-dependent methods. It is as yet unknown if silver-exposed bacteria can enter a metabolically active but non-culturable state. In this study, the efficacy of chemically synthesized AgNPs and silver as silver nitrate (AgNO3) against planktonic cells and biofilms of Pseudomonas aeruginosa AdS was investigated in microtiter plate assays, using cultural as well as culture-independent methods. In liquid medium, AgNPs and AgNO3 inhibited both planktonic growth and biofilm formation. The efficacy of AgNPs and AgNO3 against established, 24 h-old biofilms and planktonic stationary-phase cells was compared by exposure to silver in deionized water. Loss of culturability of planktonic cells was always higher than that of the attached biofilms. However, resuspended biofilm cells became more susceptible to AgNPs and AgNO3 than attached biofilms. Thus, the physical state of bacteria within biofilms rendered them more tolerant to silver compared with the planktonic state. Silver-exposed cells that had become unculturable still displayed signs of viability: they contained rRNA, determined by fluorescent in situ hybridization, as an indicator for potential protein synthesis, maintained their membrane integrity as monitored by differential live/dead staining, and displayed significant levels of adenosine triphosphate. It was concluded that AgNPs and AgNO3 in concentrations at which culturability was inhibited, both planktonic and biofilm cells of P. aeruginosa were still intact and metabolically active, reminiscent of the viable but non-culturable state known to be induced in pathogenic bacteria in response to stress conditions. This observation is important for a realistic assessment of the antimicrobial properties of AgNPs.

Lesser-known or hidden reservoirs of infection and implications for adequate prevention strategies: Where to look and what to look for.

In developing hygiene strategies, in recent years, the major focus has been on the hands as the key route of infection transmission. However, there is a multitude of lesser-known and underestimated reservoirs for microorganisms which are the triggering sources and vehicles for outbreaks or sporadic cases of infection. Among those are water reservoirs such as sink drains, fixtures, decorative water fountains and waste-water treatment plants, frequently touched textile surfaces such as private curtains in hospitals and laundry, but also transvaginal ultrasound probes, parenteral drug products, and disinfectant wipe dispensers. The review of outbreak reports also reveals Gram-negative and multiple-drug resistant microorganisms to have become an increasingly frequent and severe threat in medical settings. In some instances, the causative organisms are particularly difficult to identify because they are concealed in biofilms or in a state referred to as viable but nonculturable, which eludes conventional culture media-based detection methods. There is an enormous preventative potential in these insights, which has not been fully tapped. New and emerging pathogens, novel pathogen detection methods, and hidden reservoirs of infection should hence be given special consideration when designing the layout of buildings and medical devices, but also when defining the core competencies for medical staff, establishing programmes for patient empowerment and education of the general public, and when implementing protocols for the prevention and control of infections in medical, community and domestic settings.

Minimum information about a biofilm experiment (MIABiE): standards for reporting experiments and data on sessile microbial communities living at interfaces.

The minimum information about a biofilm experiment (MIABiE) initiative has arisen from the need to find an adequate and scientifically sound way to control the quality of the documentation accompanying the public deposition of biofilm-related data, particularly those obtained using high-throughput devices and techniques. Thereby, the MIABiE consortium has initiated the identification and organization of a set of modules containing the minimum information that needs to be reported to guarantee the interpretability and independent verification of experimental results and their integration with knowledge coming from other fields. MIABiE does not intend to propose specific standards on how biofilms experiments should be performed, because it is acknowledged that specific research questions require specific conditions which may deviate from any standardization. Instead, MIABiE presents guidelines about the data to be recorded and published in order for the procedure and results to be easily and unequivocally interpreted and reproduced. Overall, MIABiE opens up the discussion about a number of particular areas of interest and attempts to achieve a broad consensus about which biofilm data and metadata should be reported in scientific journals in a systematic, rigorous and understandable manner.