Forced Wetting Properties of Bacteria-Laden Droplets Experiencing Initial Evaporation.

Microbial adhesion and spreading on surfaces are crucial aspects in environmental and industrial settings being also the early stage of complex surface-attached microbial communities known as biofilms. In this work, Pseudomonas fluorescens-laden droplets on hydrophilic substrates (glass coupons) are allowed to partially evaporate before running wetting measurements, to study the effect of evaporation on their interfacial behavior during spillover or splashing. Forced wetting is investigated by imposing controlled centrifugal forces, using a novel rotatory device (Kerberos). At a defined evaporation time, results for the critical tangential force required for the inception of sliding are presented. Microbe-laden droplets exhibit different wetting/spreading properties as a function of the imposed evaporation times. It is found that evaporation is slowed down in bacterial droplets with respect to nutrient medium ones. After sufficient drying times, bacteria accumulate at droplet edges, affecting the droplet shape and thus depinning during forced wetting tests. Droplet rear part does not pin during the rotation test, while only the front part advances and spreads along the force direction. Quantitative results obtained from the well-known Furmidge's equation reveal that force for sliding inception increases as evaporation time increases. This study can be of support for control of biofilm contamination and removal and possible design of antimicrobial/antibiofouling surfaces.

In vitro and in silico evaluation of the serrapeptase effect on biofilm and amyloids of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an emerging threat for hospitalized and cystic fibrosis patients. Biofilm, a microbial community embedded in extracellular polymeric substance, fortifies bacteria against the immune system. In biofilms, the expression of functional amyloids is linked with highly aggregative, multi-resistant strains, and chronic infections. Serrapeptase (SPT), a protease possessing similar or superior anti-microbial properties with many antibiotics, presents anti-amyloid potential. However, studies on the employment of SPT against Pseudomonas biofilms and Fap amyloid, or the possible mechanisms of action are scarce. Here, SPT inhibited biofilm formation of P. aeruginosa ATCC 27853 on both plastic and glass surfaces, with an IC50 of 11.26 µg/mL and 0.27 µg/mL, respectively. The inhibitory effect of SPT on biofilm was also verified with optical microscopy of crystal violet-stained biofilms and with confocal microscopy. Additionally, SPT caused a dose-dependent decrease of bacterial viability (IC50 of 3.07 µg/mL) as demonstrated by MTT assay. Reduction of bacterial functional amyloids was also demonstrated, employing both fluorescence microscopy with thioflavin T and photometrical determination of Congo-red-positive compounds. Both viability and functional amyloids correlated significantly with biofilm inhibition. Finally, in silico molecular docking studies provided a mechanistic insight into the interaction of SPT with FapC or FapD, proving that both peptides are possible targets of SPT. These results offer new insights into the biofilm formation of P. aeruginosa and potentiate the involvement of SPT in the prevention and eradication of Pseudomonas biofilms. KEY POINTS: • Serrapeptase inhibits biofilm formation of P. aeruginosa on plastic and glass. • Biofilm inhibition correlated with reduced viability and functional amyloid levels. • In silico studies indicated that serrapeptase may target FapC and FapD peptides.

Wetting and Imbibition Characteristics of Pseudomonas fluorescens Biofilms Grown on Stainless Steel.

This study aims to provide insights into biofilm resistance associated with their structural properties acquired during formation and development. On this account, the wetting and imbibition behavior of dehydrated Pseudomonas fluorescens biofilms grown on stainless steel electropolished substrates is thoroughly examined at different biofilm ages. A polar liquid (water) and a non-polar liquid (diiodomethane) are employed as wetting agents in the form of sessile droplets. A mathematical model is applied to appraise the wetting and imbibition performance of biofilms incorporating the evaporation of sessile droplets. The present results show that the examined biofilms are hydrophilic. The progressive growth of biofilms leads to a gradual increase of substrate surface coverage─up to full coverage─accompanied by a gradual decrease of biofilm surface roughness. It is noteworthy that just after 24 h of biofilm growth, the surface roughness increases about 6.7 times the roughness of the clean stainless steel surface. It is further found that the imbibition of liquid in the biofilm matrix is restricted only to the biofilm region under the sessile droplet. The lack of further capillary imbibition into the biofilm structure, beyond the droplet deposition region, implies that the biofilm matrix is not in the form of an extended network of interconnected micro/nanopores. All in all, the present results indicate a resilient biofilm structure to biocide penetration despite its hydrophilic nature.

Wetting of Dehydrated Hydrophilic Pseudomonas fluorescens Biofilms under the Action of External Body Forces.

Wetting of dehydrated Pseudomonas fluorescens biofilms grown on glass substrates by an external liquid is employed as a means to investigate the complex morphology of these biofilms along with their capability to interact with external fluids. The porous structure left behind after dehydration induces interesting droplet spreading on the external surface and imbibition into pores upon wetting. Static contact angles and volume loss by imbibition measured right upon droplet deposition indicate that biofilms of higher incubation times show a higher porosity and effective hydrophilicity. Furthermore, during subsequent rotation tests, using Kerberos device, these properties dictate a peculiar forced wetting/spreading behavior. As rotation speed increases a long liquid tail forms progressively at the rear part of the droplet, which stays pinned at all times, while only the front part of the droplet depins and spreads. Interestingly, the experimentally determined retention force for the onset of droplet sliding on biofilm external surface is lower than that on pure glass. An effort is made to describe such complex forced wetting phenomena by presenting apparent contact angles, droplet length, droplet shape contours, and edges position as obtained from detailed image analysis.

Implications of handling practices on the ecotoxic profile of alumina nanoparticles towards the bacteria Vibrio fischeri.

The complex nature and behavior of Engineered Nanomaterials (ENMs) has led to adoption of customized experimental ecotoxicity practices that are prone to possible artefacts in the inherent toxic properties of ENMs. In addition, the lack of standardized handling procedures for the ecotoxicity testing of ENMs prevents the development of experimental protocols for regulatory purposes. In this study, a suite of techniques for dispersion of ENMs was adopted and tested for two types of surface-modified alumina nanoparticles-one hydrophobic and one hydrophilic-towards the bacteria, Vibrio fischeri. The effect of certain handling practices on the observed ecotoxic effects on V. fischeri was examined. The overall goal was to evaluate by what means the handling practices of ENMs may affect the obtained toxicity results. It was realized that the toxicity of the hydrophilic and hydrophobic ENMs was mainly affected by the centrifugation and the salinity of the tested dispersions, respectively. It is more likely that both aluminium and coating substance contributed to the overall toxicity. Toxicity results are discussed with regard to generic physicochemical characteristics of the dispersions.

The role of air relative humidity on the wettability of Pseudomonas fluorescens AR11 biofilms.

Biofilms are complex porous materials formed by microorganisms, polysaccharides, proteins, eDNA, inorganic matter, and water. They are ubiquitous in various environmental niches and are known to grow at solid-liquid, solid-air and air-liquid interfaces, often causing problems in several industrial and sanitary fields. Their removal is a challenge in many applications and numerous studies have been conducted to identify promising chemical species as cleaning agents. While these substances target specific components of biofilm structure, the role of water content in biofilm, and how it can influence wettability and detergent absorption have been quite neglected in the literature. Estimating water content in biofilm is a challenging task due to its heterogeneity in morphology and chemical composition. In this study, we controlled water content in Pseudomonas fluorescens AR 11 biofilms grown on submerged glass slides by regulating environmental relative humidity after drying. Interfacial properties of biofilm were investigated by measuring wetting of water and soybean oil. The morphology of biofilm structure was evaluated using Confocal Laser Scanning Microscopy and Scanning Electron Microscopy. The results showed that biofilm water content has a significant and measurable effect on its wettability, leading to the hypothesis that a preliminary control of water content can play a crucial role in biofilm removal process.

Hexavalent chromium release from lignite fly ash and related ecotoxic effects.

Hexavalent chromium Cr(VI) is a pollutant of immense concern due to its high mobility to water sources and highly toxic properties. In most cases, Cr(VI) could be released from lignite fly ash in aquatic environment when fly ash comes into contact with water. In this study, the contribution of the leaching patterns and bioavailability of Cr(VI) from lignite fly ash to the overall ecotoxic properties of fly ash leachates was originally examined and leaching procedures were evaluated in this context. A series of customized leaching tests were conducted and a battery of ecotoxicity tests including the crustacean Daphnia magna and the photobacterium Vibrio fischeri was applied. The leaching of Cr(VI) was pH and liquid to solid (L/S) ratio dependent, exhibiting the highest releases at pH values between 7 and 8. At the liquid to solid ratio (L/S) equal to 100 L/kg, the (CrVI) release reached a plateau, implying the presence of diffusion constrains and/or solubility hindrances. The toxic effect of the leachates obtained under leaching at pH 7 towards D. magna was relatively high (TU = 28.6 (23.8-35.7) at L/S = 10 L/kg). Interestingly, the toxicity of the leachates towards D. magna not only was significantly correlated to Cr(VI) (r = 0.961, P < 0.01), but the toxicity of the leachates (in absolute values) was matching the toxicity of the Cr(VI) revealing its remarkable contribution to the overall effect. In addition, the lower sensitivity of the bacteria V. fischeri when exposed to the leachates, along with the time dependence of the toxicity profiles supported the interpretation of the results obtained in this study.

Wetting properties of dehydrated biofilms under different growth conditions.

Biofilms are resilient to environmental conditions and often resistant even to strong disinfectants. It is crucial to investigate their interfacial properties, which can be effectively characterized by wetting analysis. Wetting phenomena on biofilm surfaces have been poorly investigated in literature, in particular a systematic study of wetting on real biofilm-coated substrates including the application of external body forces (forced wetting, i.e.: centrifugal and gravitational forces) is missing. The aim of this work is to study the role of nutrient and shear flow conditions on wetting properties of Pseudomonas fluorescens dehydrated biofilms, grown on glass substrates. An innovative device (Kerberos®), capable to study spreading/sliding behavior under the application of external body forces, is used here for a systematic analysis of wetting/de-wetting liquid droplets on horizontal substrates under the action of tangential forces. Results prove that, under different growth conditions, (i.e., nutrients and imposed flow), biofilms exhibit different wetting properties. At lower nutrient/shear flow conditions, biofilms show spreading/sliding behavior close to that of pure glass. At higher nutrient and shear flow conditions, droplets on biofilms show spreading followed by imbibition soon after deposition, which leads to peculiar droplet depinning during the rotation test. Wetting properties are derived as a function of the rotation speed from both top and side views videoframes through a dedicated image analysis technique. A detailed analysis of biofilm formation and morphology/topography is also provided here.

SARS-CoV-2 adsorption on suspended solids along a sewerage network: mathematical model formulation, sensitivity analysis, and parametric study.

Accounting for SARS-CoV-2 adsorption on solids suspended in wastewater is a necessary step towards the reliable estimation of virus shedding rate in a sewerage system, based on measurements performed at a terminal collection station, i.e., at the entrance of a wastewater treatment plant. This concept is extended herein to include several measurement stations across a city to enable the estimation of spatial distribution of virus shedding rate. This study presents a pioneer general model describing the most relevant physicochemical phenomena with a special effort to reduce the complicated algebra. This is performed both in the topology regime, introducing a discrete-continuous approach, and in the domain of independent variables, introducing a monodisperse moment method to reduce the dimensionality of the resulting population balance equations. The resulting simplified model consists of a large system of ordinary differential equations. A sensitivity analysis is performed with respect to some key parameters for a single pipe topology. Specific numerical techniques are employed for the integration of the model. Finally, a parametric case study for an indicative-yet realistic-sewerage piping system is performed to show how the model is applied to SARS-CoV-2 adsorption on wastewater solids in the presence of other competing species. This is the first model of this kind appearing in scientific literature and a first step towards setting up an inverse problem to assess the spatial distribution of virus shedding rate based on its concentration in wastewater.

SARS-CoV-2 wastewater monitoring using a novel PCR-based method rapidly captured the Delta-to-Omicron ΒΑ.1 transition patterns in the absence of conventional surveillance evidence.

Conventional SARS-CoV-2 surveillance based on genotyping of clinical samples is characterized by challenges related to the available sequencing capacity, population sampling methodologies, and is time, labor, and resource-demanding. Wastewater-based variant surveillance constitutes a valuable supplementary practice, since it does not require extensive sampling, and provides information on virus prevalence in a timely and cost-effective manner. Consequently, we developed a sensitive real-time RT-PCR-based approach that exclusively amplifies and quantifies SARS-CoV-2 genomic regions carrying the S:Δ69/70 deletion, indicative of the Omicron BA.1 variant, in wastewater. The method was incorporated in the analysis of composite daily samples taken from the main Wastewater Treatment Plant of Thessaloniki, Greece, from 1 December 2021. The applicability of the methodology is dependent on the epidemiological situation. During Omicron BA.1 global emergence, Thessaloniki was experiencing a massive epidemic wave attributed solely to the Delta variant, according to genomic surveillance data. Since Delta does not possess the S:Δ69/70, the emergence of Omicron BA.1 could be monitored via the described methodology. Omicron BA.1 was detected in sewage samples on 19 December 2021 and a rapid increase of its viral load was observed in the following 10-day period, with an estimated early doubling time of 1.86 days. The proportion of the total SARS-CoV-2 load attributed to BA.1 reached 91.09 % on 7 January, revealing a fast Delta-to-Omicron transition pattern. The detection of Omicron BA.1 subclade in wastewater preceded the outburst of reported (presumable) Omicron cases in the city by approximately 7 days. The proposed wastewater surveillance approach based on selective PCR amplification of a genomic region carrying a deletion signature enabled rapid, real-time data acquisition on Omicron BA.1 prevalence and dynamics during the slow remission of the Delta wave. Timely provision of these results to State authorities readily influences the decision-making process for targeted public health interventions, including control measures, awareness, and preparedness.

The COVID-19 pandemic as inspiration to reconsider epidemic models: A novel approach to spatially homogeneous epidemic spread modeling.

Epidemic spread models are useful tools to study the spread and the effectiveness of the interventions at a population level, to an epidemic. The workhorse of spatially homogeneous class models is the SIR-type ones comprising ordinary differential equations for the unknown state variables. The transition between different states is expressed through rate functions. Inspired by -but not restricted to- features of the COVID-19 pandemic, a new framework for modeling a disease spread is proposed. The main concept refers to the assignment of properties to each individual person as regards his response to the disease. A multidimensional distribution of these properties represents the whole population. The temporal evolution of this distribution is the only dependent variable of the problem. All other variables can be extracted by post-processing of this distribution. It is noteworthy that the new concept allows an improved consideration of vaccination modeling because it recognizes vaccination as a modifier of individuals response to the disease and not as a means for individuals to totally defeat the disease. At the heart of the new approach is an infection age model engaging a sharp cut-off. This model is analyzed in detail, and it is shown to admit self-similar solutions. A hierarchy of models based on the new approach, from a generalized one to a specific one with three dominant properties, is derived. The latter is implemented as an example and indicative results are presented and discussed. It appears that the new framework is general and versatile enough to simulate disease spread processes and to predict the evolution of several variables of the population during this spread.

Detecting SARS-CoV-2 lineages and mutational load in municipal wastewater and a use-case in the metropolitan area of Thessaloniki, Greece.

The COVID-19 pandemic represents an unprecedented global crisis necessitating novel approaches for, amongst others, early detection of emerging variants relating to the evolution and spread of the virus. Recently, the detection of SARS-CoV-2 RNA in wastewater has emerged as a useful tool to monitor the prevalence of the virus in the community. Here, we propose a novel methodology, called lineagespot, for the monitoring of mutations and the detection of SARS-CoV-2 lineages in wastewater samples using next-generation sequencing (NGS). Our proposed method was tested and evaluated using NGS data produced by the sequencing of 14 wastewater samples from the municipality of Thessaloniki, Greece, covering a 6-month period. The results showed the presence of SARS-CoV-2 variants in wastewater data. lineagespot was able to record the evolution and rapid domination of the Alpha variant (B.1.1.7) in the community, and allowed the correlation between the mutations evident through our approach and the mutations observed in patients from the same area and time periods. lineagespot is an open-source tool, implemented in R, and is freely available on GitHub and registered on bio.tools.

The role of flow in bacterial biofilm morphology and wetting properties.

Biofilms are bacterial communities embedded in an extracellular matrix, able to adhere to surfaces. Different experimental set-ups are widely used for in vitro biofilm cultivation; however, a well-defined comparison among different culture conditions, especially suited to interfacial characterization, is still lacking in the literature. The main objective of this work is to study the role of flow on biofilm formation, morphology and interfacial properties. Three different in vitro setups, corresponding to stagnant, shaking, and laminar flow conditions (custom-made flow cell), are used in this work to grow single strain biofilms of Pseudomonas fluorescens AR 11 on glass coupons. Results show that flow conditions significantly influenced biofilm formation kinetics, affecting mass transfer and cell attachment/detachment processes. Distinct morphological patterns are found under different flow regimes. Static contact angle data do not depend significantly on biofilm growth conditions in the parametric range investigated in this work.