The in situ Production of Aquatic Fluorescent Organic Matter in a Simulated Freshwater Laboratory Model.

Dissolved organic matter (DOM) is ubiquitous throughout aquatic systems. Fluorescence techniques can be used to characterize the fluorescing proportion of DOM, aquatic fluorescent organic matter (AFOM). AFOM is conventionally named in association with specific fluorescence "peaks," which fluoresce in similar optical regions as microbially-derived proteinaceous material (Peak T), and terrestrially-derived humic-like compounds (Peaks C/C+), with Peak T previously being investigated as a tool for bacterial enumeration within freshwaters. The impact of anthropogenic nutrient loading on the processing of DOM by microbial communities is largely unknown. Previous laboratory studies utilizing environmental freshwater have employed growth media with complex background fluorescence, or very high nutrient concentrations, preventing the investigation of AFOM production under a range of more representative nutrient concentrations within a matrix exhibiting very low background fluorescence. We describe a laboratory-based model with Pseudomonas aeruginosa that incorporates a low fluorescence growth matrix consisting of a simulated freshwater (SFW), representative of low-hardness freshwater systems allowing controlled nutrient conditions to be studied. The effects of microbial processing of DOM as a function of available nitrogen, phosphorous, and dissolved organic carbon (DOC) in the form of glucose were investigated over 48 h at highly resolved time increments. The model system demonstrates the production of a range of complex AFOM peaks in the presence and absence of DOC, revealing no linear relationship between cell numbers and any of the peaks for the bacterial species studied, with AFOM peaks increasing with microbial cell number, ranging from 55.2 quinine sulfate units (QSU) per 106 cells to 155 QSU per 106 cells (p < 0.05) for Peak T during the exponential growth phase of P. aeruginosa under high nutrient conditions with 5 mg L-1 DOC. Nutrient and DOC concentration was found to cause differential production of autochthonous- or allochthonous-like AFOM, with lower DOC concentrations resulting in higher Peak T production relative to Peaks C/C+ upon the addition of nutrients, and high DOC concentrations resulting in higher Peak C/C+ production relative to Peak T. Our results show the production of allochthonous-like AFOM from a simple and non-fluorescent carbon source, and provide uncertainty in the use of Peak T as a reliable surrogate for specific bacterial enumeration, particularly in dynamic or nutrient-impacted environments, pointing toward the use of fluorescence as an indicator for microbial metabolism.

A systematic approach to understand hydrogeochemical dynamics in large river systems: Development and application to the River Ganges (Ganga) in India.

Large river systems, such as the River Ganges (Ganga), provide crucial water resources for the environment and society, yet often face significant challenges associated with cumulative impacts arising from upstream environmental and anthropogenic influences. Understanding the complex dynamics of such systems remains a major challenge, especially given accelerating environmental stressors including climate change and urbanization, and due to limitations in data and process understanding across scales. An integrated approach is required which robustly enables the hydrogeochemical dynamics and underpinning processes impacting water quality in large river systems to be explored. Here we develop a systematic approach for improving the understanding of hydrogeochemical dynamics and processes in large river systems, and apply this to a longitudinal survey (> 2500 km) of the River Ganges (Ganga) and key tributaries in the Indo-Gangetic basin. This framework enables us to succinctly interpret downstream water quality trends in response to the underpinning processes controlling major element hydrogeochemistry across the basin, based on conceptual water source signatures and dynamics. Informed by a 2019 post-monsoonal survey of 81 river bank-side sampling locations, the spatial distribution of a suite of selected physico-chemical and inorganic parameters, combined with segmented linear regression, reveals minor and major downstream hydrogeochemical transitions. We use this information to identify five major hydrogeochemical zones, characterized, in part, by the inputs of key tributaries, urban and agricultural areas, and estuarine inputs near the Bay of Bengal. Dominant trends are further explored by investigating geochemical relationships (e.g. Na:Cl, Ca:Na, Mg:Na, Sr:Ca and NO3:Cl), and how water source signatures and dynamics are modified by key processes, to assess the relative importance of controls such as dilution, evaporation, water-rock interactions (including carbonate and silicate weathering) and anthropogenic inputs. Mixing/dilution between sources and water-rock interactions explain most regional trends in major ion chemistry, although localized controls plausibly linked to anthropogenic activities are also evident in some locations. Temporal and spatial representativeness of river bank-side sampling are considered by supplementary sampling across the river at selected locations and via comparison to historical records. Limitations of such large-scale longitudinal sampling programs are discussed, as well as approaches to address some of these inherent challenges. This approach brings new, systematic insight into the basin-wide controls on the dominant geochemistry of the River Ganga, and provides a framework for characterising dominant hydrogeochemical zones, processes and controls, with utility to be transferable to other large river systems.

Real-time detection of volatile metabolites enabling species-level discrimination of bacterial biofilms associated with wound infection.

AIMS: The main aim of this study was to investigate the real-time detection of volatile metabolites for the species-level discrimination of pathogens associated with clinically relevant wound infection, when grown in a collagen wound biofilm model. METHODS AND RESULTS: This work shows that Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes produce a multitude of volatile compounds when grown as biofilms in a collagen-based biofilm model. The real-time detection of these complex volatile profiles using selected ion flow tube mass spectrometry and the use of multivariate statistical analysis on the resulting data can be used to successfully differentiate between the pathogens studied. CONCLUSIONS: The range of bacterial volatile compounds detected between the species studied vary and are distinct. Discrimination between bacterial species using real-time detection of volatile metabolites and multivariate statistical analysis was successfully demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of rapid point-of-care diagnostics for wound infection would improve diagnosis and patient care. Such technological approaches would also facilitate the appropriate use of antimicrobials, minimizing the emergence of antimicrobial resistance. This study further develops the use of volatile metabolite detection as a new diagnostic approach for wound infection.

Laboratory In-Situ Production of Autochthonous and Allochthonous Fluorescent Organic Matter by Freshwater Bacteria.

This work investigates the origin and range of fluorescent organic matter (FOM) produced in-situ by environmentally sourced freshwater bacteria. Aquatic FOM is an essential component in global carbon cycling and is generally classified as either autochthonous, produced in-situ via microbial processes, or allochthonous, transported into aquatic systems from external sources. We have demonstrated that, within laboratory model systems, environmentally sourced mixed microbial communities and bacterial isolates can produce and/or export FOM associated with both autochthonous and allochthonous material. This study focuses on fluorescence peak B, T, M, C and C+, exploring (1) the cellular nature of FOM produced, (2) FOM exported as extracellular material into the water column and (3) the impact of physical cell lysis on FOM signature. For the laboratory model systems studied, Peak T fluorescence is retained within bacterial cells (>68%), while Peak C fluorescence is mainly observed as extracellular material (>80%). Peak M is identified as both cellular and extracellular FOM, produced by all isolated freshwater microorganisms investigated. The origin of Peak C+ is postulated to originate from functional metabolites associated with specific microorganisms, seen specifically within the Pseudomonas sp. monoculture here. This work challenges the binary classification of FOM as either allochthonous or autochthonous, suggesting that FOM processing and production occurs along a dynamic continuum. Within this study, fluorescence intensity data for the environmental bacteria isolate monocultures are presented as enumeration corrected data, for the first time providing quantitative fluorescence data per bacterial colony forming unit (cfu). From this, we are able to assess the relative contribution of different bacteria to the autochthonous FOM pool and if this material is cellular or extracellular.

An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics.

BACKGROUND: The majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time. RESULTS: The collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide- 1 was achieved by 30 h of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2-1.6 log reduction in biofilm density at 72 h compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 h at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation. CONCLUSIONS: The collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies.

Application of bacterial bioluminescence to assess the efficacy of fast-acting biocides.

Traditional microbiological techniques are used to provide reliable data on the rate and extent of kill for a range of biocides. However, such techniques provide very limited data regarding the initial rate of kill of fast-acting biocides over very short time domains. This study describes the application of a recombinant strain of Escherichia coli expressing the Photorhabdus luminescens lux operon as a whole-cell biosensor. Light emission is linked directly to bacterial metabolism; therefore, by monitoring light output, the impact of fast-acting biocides can be assessed. Electrochemically activated solutions (ECASs), bleach, Virkon, and ethanol were assessed at three concentrations (1%, 10%, 80%) in the presence of organic soiling. Over a 2-s time course, 80% ECAS produced the greatest reduction in light output in the absence of organic load but was strongly inhibited by its presence. Eighty percent ethanol outperformed all tested biocides in the presence of organic soil. Bleach and Virkon produced similar reductions in bioluminescence at matched concentrations within the time course of the assay. It was also demonstrated that the assay can be used to rapidly assess the impact of organic soiling. The use of bioluminescent bacteria as whole-cell bioreporters allows assessment of the relative efficacies of fast-acting biocides within milliseconds of application. The assay can be used to investigate activity over short or extended time domains to confirm complete metabolic inhibition of the bioreporter. Moreover, the assay may enable further elucidation of their mechanism of action by allowing the investigation of activity over time domains precluded by traditional microbiology.

In vitro diffusion bed, 3-day repeat challenge 'capacity' test for antimicrobial wound dressings.

The aim of this study was to develop an in vitro wound infection model that allows the comparison of the bacterial kill rate of antimicrobial wound dressings over the course of 3 days, with renewed microbial challenges each day, under realistic wound-like conditions. A test bed model of a moderately exuding wound was constructed from a hydrogel containing releasable foetal calf serum (FCS), and cellulose discs dosed with test microbes (Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa) suspended in 50% FCS applied at the interface between the test dressing and the hydrogel test bed. Freshly prepared discs were used to challenge the same dressing over a 23-hour period for a course of 3 days. Different test dressings produced differing kill rates, allowing quantitative comparison of both their immediate activity and their capacity to continue working over 3 days, within a fluid-donating system similar to the situation in vivo. The reported method has significant advantages over established test procedures since it enables the researcher to assess the antimicrobial capacity of wound dressings to continue working under conditions that match those encountered in wounds. These key conditions are those that would be expected to impede the action of the dressing and protect the infecting organisms.