Vibrio fischeri: a model for host-associated biofilm formation.

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

Lighting the way: how the Vibrio fischeri model microbe reveals the complexity of Earth's "simplest" life forms.

Vibrio (Aliivibrio) fischeri's initial rise to fame derived from its alluring production of blue-green light. Subsequent studies to probe the mechanisms underlying this bioluminescence helped the field discover the phenomenon now known as quorum sensing. Orthologs of quorum-sensing regulators (i.e., LuxR and LuxI) originally identified in V. fischeri were subsequently uncovered in a plethora of bacterial species, and analogous pathways were found in yet others. Over the past three decades, the study of this microbe has greatly expanded to probe the unique role of V. fischeri as the exclusive symbiont of the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Buoyed by this optically amenable host and by persistent and insightful researchers who have applied novel and cross-disciplinary approaches, V. fischeri has developed into a robust model for microbe-host associations. It has contributed to our understanding of how bacteria experience and respond to specific, often fluxing environmental conditions and the mechanisms by which bacteria impact the development of their host. It has also deepened our understanding of numerous microbial processes such as motility and chemotaxis, biofilm formation and dispersal, and bacterial competition, and of the relevance of specific bacterial genes in the context of colonizing an animal host. Parallels in these processes between this symbiont and bacteria studied as pathogens are readily apparent, demonstrating functional conservation across diverse associations and permitting a reinterpretation of "pathogenesis." Collectively, these advances built a foundation for microbiome studies and have positioned V. fischeri to continue to expand the frontiers of our understanding of the microbial world inside animals.

A prototrophic suppressor of a Vibrio fischeri D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >1011 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative broad-spectrum racemase of V. fischeri, bsrF. An engineered bsrF allele lacking the putative secretion signal (ΔSS-bsrF) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS-bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS-bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance. IMPORTANCE: D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.

A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

Corrected and republished from: "Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid".

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

Solar Photodegradation of a Novel des-F(6)-Fluoroquinolone, Garenoxacin, and Ecotoxicity of Its Phototransformation Products.

Garenoxacin (GRNX) is a novel des-F(6)-fluoroquinolone on the horizon; thus, its fate and risk in the aquatic environment deserve attention. This study systematically investigated, for the first time, the phototransformation of GRNX under simulated and natural sunlight and assessed the ecotoxicity of its photodegradation products. Phototransformation of GRNX was observed to depend strongly on its ionization state, with direct photolysis and self-sensitized photolysis having comparable contributions for the cationic and zwitterionic species, while the latter dominated for the anionic species. Singlet oxygen generated via the self-sensitized photolysis of GRNX was the major reactive oxygen species in its photodegradation. Phototransformation of GRNX in different ionization states followed distinct pathways, with defluorination of the difluoromethyl group occurring only for the zwitterionic and anionic species. GRNX photodegradation in natural water could be described by a simple kinetic model based on the measured steady-state concentrations of 1O2 and ·OH. Toxicity tests with Vibrio fischeri and Chlorella vulgaris consistently indicate that the generation of hydroxylation and decarboxylation products during photodegradation of GRNX increased the acute toxicity. These findings not only provide insights into the fate of GRNX in sunlit surface water but also reveal the potentially significant risk of its photodegradation products to the aquatic ecosystem.

Ecotoxicological assessment of cyclic peptides produced by a Planktothrix rubescens bloom: Impact on aquatic model organisms.

Cyanobacterial blooms, a natural phenomenon in freshwater ecosystems, have increased in frequency and severity due to climate change and eutrophication. Some cyanobacteria are able to produce harmful substances called cyanotoxins. These metabolites possess different chemical structures and action mechanisms representing a serious concern for human health and the environment. The most studied cyanotoxins belong to the group of microcystins which are potent hepatotoxins. Anabaenopeptins are another class of cyclic peptides produced by certain species of cyanobacteria, including Planktothrix spp. Despite limited knowledge regarding individual effects of anabaenopeptins on freshwater organisms, reports have identified in vivo toxicity in representatives of freshwater zooplankton by cyanobacterial extracts or mixtures containing anabaenopeptins. This study focused on the isolation and toxicity evaluation of the cyanotoxins produced in the 2022 Planktothrix rubescens bloom in Averno lake, Italy. The three main cyclic peptides have been isolated and identified by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and optical analyses as anabaenopeptins A and B, and oscillamide Y. Ecotoxicological tests on the aquatic model organisms Daphnia magna (crustacean), Raphidocelis subcapitata (algae), and Aliivibrio fischeri (bacterium) revealed that anabaenopeptins A and B do not generate significant toxicity at environmentally relevant concentrations, being also found a stimulatory effect on R. subcapitata in the case of anabaenopeptin A. By contrast, oscillamide Y displayed toxicity. Ecological implications based on ECOSAR predictions align with experimental data. Moreover, long-term exposure bioassays on different green unicellular algae species showed that R. subcapitata was not significantly affected, while Scenedesmus obliquus and Chlorella vulgaris exhibited altered growth patterns. These results, together with the already-known background in literature, highlight the complexity of interactions between organisms and the tested compounds, which may be influenced by species-specific sensitivities, physiological differences, and modes of action, possibly affected by parameters like lipophilicity.

Physicochemical and ecotoxicological approaches for Moknine Continental Sebkha in Tunisia.

Degradation of water quality is an emerging issue in many developing countries. In this context, industrial and domestic effluents heavily contaminate the coast of Moknine Continental Sebkha in Tunisia. The present study aimed to biomonitor the seawater quality of the Moknine Continental Sebkha coast using physicochemical and ecotoxicological approaches. The ecotoxicological assessment was performed using three species representing different trophic levels, namely Vibrio fischeri, Selenastrum capricornutum, and Lepidium sativum. In the physicochemical analysis such as BOD (biochemical oxygen demand), COD (chemical oxygen demand), TSS (total suspended solids), TOC (total organic carbon), NO3- (nitrate), AOX (adsorbable organic halogen), the recorded levels of pH and total suspended solids did not comply with the Tunisian standard (NT.09.11/1983). The ecotoxicological data confirmed that the tested water samples displayed toxicity to two test indicators L. sativum and S. capricornutum. A targeted chemical screening of the Moknine Continental Sebkha coast previously performed revealed the presence of total mercury, four phthalate acid esters, and one non-phthalate plasticizer, a fact that could explain the observed ecotoxicological effects and therefore might harm the biotic area and the health of the surrounding population.

Effect of zeolites on the reduction of the ecotoxicity of carbamazepine in the environment.

In this study, the impact of calcination of zeolites on the ecotoxicity of carbamazepine solutions in two matrices, water and synthetic sewage, was assessed. Two types of zeolites were tested: natural zeolite, in the form of a zeolite rock consisting mainly of clinoptilolite, and a synthetic zeolite type 5 A. Additionally, zeolites were calcined at a temperature of 200 °C. The kinetics of carbamazepine adsorption in aqueous solutions and in synthetic sewage matrix was determined. Higher adsorption capacity was obtained for carbamazepine aqueous solutions as well as zeolites after the calcination process. Considering type of zeolite, the highest and fastest uptake of carbamazepine was observed for natural zeolite after calcination. In the case of ecotoxicity, carbamazepine solutions before adsorption was the most toxic towards Raphidocelis subcapitata, next Aliivibrio fischeri and Daphnia magna, regardless to the matrix type. The differentiation in toxicity regarding the type of matrix was observed, in the case of algae and bacteria, higher toxicity was demonstrated by carbamazepine solutions in the water matrix, while in the case of crustaceans-the sewage matrix. After the adsorption process, the toxicity of carbamazepine solutions on zeolites decreased by 34.5-60.9 % for R. subcapitata, 33-39 % for A. fischeri and 55-60 % for D. magna, thus confirming the effectiveness of the proposed method of carbamazepine immobilization.

Long-Chain Alkylthio Cyclodextrin Derivatives for Modulation of Quorum-Sensing-Based Bioluminescence in Aliivibrio fischeri Model System.

Quorum sensing (QS) allows bacteria to coordinate their activities by producing and detecting low-molecular-weight signal molecules based on population density, thereby controlling the infectivity of bacteria through various virulence factors. Quorum-sensing inhibition is a promising approach to tackle bacterial communication. Cyclodextrins (CDs) are a class of cyclic oligosaccharides that reversibly encapsulate the acyl chain of the signal molecules, thereby preventing their binding to receptors and interrupting bacterial communication. This results in the inhibition of the expression of various properties, including different virulence factors. To examine the potential quorum-quenching (QQ) ability of newly prepared cyclodextrin derivatives, we conducted short-term tests using Aliivibrio fischeri, a heterotrophic marine bacterium capable of bioluminescence controlled by quorum sensing. α- and ß-cyclodextrins monosubstituted with alkylthio moieties and further derivatized with quaternary ammonium groups were used as the test agents. The effect of these cyclodextrins on the quorum-sensing system of A. fischeri was investigated by adding them to an exponential growth phase of the culture and then measuring bioluminescence intensity, population growth, and cell viability. Our results demonstrate that the tested cyclodextrins have an inhibitory effect on the quorum-sensing system of A. fischeri. The inhibitory effect varies based on the length of the alkyl chain, with alkylthio substitution enhancing it and the presence of quaternary ammonium groups decreasing it. Our findings suggest that cyclodextrins can be a promising therapeutic agent for the treatment of bacterial infections.

Bioactivity of Eugenol: A Potential Antibiotic Adjuvant with Minimal Ecotoxicological Impact.

Combining commercial antibiotics with adjuvants to lower their minimum inhibitory concentration (MIC) is vital in combating antimicrobial resistance. Evaluating the ecotoxicity of such compounds is crucial due to environmental and health risks. Here, eugenol was assessed as an adjuvant for 7 commercial antibiotics against 14 pathogenic bacteria in vitro, also examining its acute ecotoxicity on various soil and water organisms (microbiota, Vibrio fischeri, Daphnia magna, Eisenia foetida, and Allium cepa). Using microdilution methods, checkerboard assays, and kinetic studies, the MICs for eugenol were determined together with the nature of its combinations with antibiotics against bacteria, some unexposed to eugenol previously. The lethal dose for the non-target organisms was also determined, as well as the Average Well Color Development and the Community-Level Physiological Profiling for soil and water microbiota. Our findings indicate that eugenol significantly reduces MICs by 75 to 98%, which means that it could be a potent adjuvant. Ecotoxicological assessments showed eugenol to be less harmful to water and soil microbiota compared to studied antibiotics. While Vibrio fischeri and Daphnia magna were susceptible, Allium cepa and Eisenia foetida were minimally affected. Given that only 0.1% of eugenol is excreted by humans without metabolism, its environmental risk when used with antibiotics appears minimal.

Influence of the chemical composition of leachates on the results of ecotoxicity tests for different slag types.

In this study, four ecotoxicological tests on Vibrio fischeri bacteria, Sinapis alba L. (white mustard), Daphnia magna S. (daphnia's) and earthworms were performed for three types of aqueous slag (ladle, blast furnace and converter) leachates with two-grain sizes (<4 mm, <10 mm). Concentrations of toxic elements and concentrations of Cr(VI), Ca, Na, Al, and other ions were determined. The raw slags were analyzed using X-ray fluorescence spectroscopy (XRFS), and major substances were determined by X-ray powder diffraction (XRD). The aqueous slag leachates passed ecotoxicological tests and met the required criteria, showing no toxicity to Vibrio fischeri and complying with white mustard test criteria. According to the results of the ecotoxicity tests with daphnia, the blast furnace slag samples were not ecotoxic, while two other slag samples were found to be entirely compliant. Characterization of the slags showed that the effect of element/ion leachability and slag grain size is essential. Biplot principal component analysis (PCA) showed that grain size does not significantly affect the separation of individuals on the plane. A positive correlation on toxicity was found with pH, conductivity, calcium content, dissolved content, salinity and fluoride concentration, whereas a negative correlation was found with magnesium concentration, dissolved organic carbon and potassium concentration. The effective concentration at 50% inhibition (EC50) value for Vibrio fischeri correlated with the first dimension of bivariate assessment. In summary, it was found that the investigated slags can be effectively reused as they comply with regulations and do not endanger the environment.

Equations for estimating binary mixture toxicity: 3-methyl-2-butanone with a series of electrophiles.

Mixture toxicity was determined for 32 binary combinations. One chemical was the non-reactive, non-polar narcotic 3-methyl-2-butanone (always chemical A) and the other was a potentially reactive electrophile (chemical B). Bioluminescence inhibition in Allovibrio fischeri was measured at 15-, 30-, and 45-minutes of exposure for A, B, and the mixture (MX). Concentration-response curves (CRCs) were developed for each chemical and used to develop predicted CRCs for the concentration addition (CA) and independent action (IA) mixture toxicity models. Also, MX CRCs were generated and compared with model predictions using the 45-minute data. Classification of observed mixture toxicity used three specific criteria: 1) predicted IA EC50 vs. CA EC50 values at 45-minutes, 2) consistency of 45-minute MX CRC fit to IA, CA, or otherwise at three effect levels (EC25, EC50 and EC75), and 3) the known/suspected mechanism of toxicity for chemical B. Mixture toxicity was then classified into one of seven groupings. As a result of the predicted IA EC50 being more toxic than the predicted CA EC50, IA represented the greater toxic hazard. For this reason, non-sham MXs having toxicity consistent with CA were classified as being "coincident" with CA rather than mechanistically-consistent with CA. Multiple linear regression analyses were performed to develop equations that can be used to estimate the toxicity of other 3M2B-containing binary mixtures. These equations were developed from the data for both IA and CA, at each exposure duration and effect level. Each equation had a coefficient of determination (r2) above 0.950 and a variance inflation factor <1.2. This approach can potentially reduce the need for mixture testing and is amenable to other model systems and to assays that evaluate toxicity at low effect levels.

Transcriptional pathways across colony biofilm models in the symbiont Vibrio fischeri.

Beneficial microbial symbionts that are horizontally acquired by their animal hosts undergo a lifestyle transition from free-living in the environment to associating with host tissues. In the model symbiosis between the Hawaiian bobtail squid and its microbial symbiont Vibrio fischeri, one mechanism used to make this transition during host colonization is the formation of biofilm-like aggregates in host mucosa. Previous work identified factors that are sufficient to induce V. fischeri biofilm formation, yet much remains unknown regarding the breadth of target genes induced by these factors. Here, we probed two widely used in vitro models of biofilm formation to identify novel regulatory pathways in the squid symbiont V. fischeri ES114. We discovered a shared set of 232 genes that demonstrated similar patterns in expression in both models. These genes comprise multiple exopolysaccharide loci that are upregulated and flagellar motility genes that are downregulated, with a consistent decrease in measured swimming motility. Furthermore, we identified genes regulated downstream of the key sensor kinase RscS that are induced independent of the response regulator SypG. Our data suggest that transcriptional regulator VpsR plays a strong role in expression of at least a subset of these genes. Overall, this study adds to our understanding of the genes involved in V. fischeri biofilm regulation while revealing new regulatory pathways branching from previously characterized signaling networks.IMPORTANCEThe V. fischeri-squid system provides an opportunity to study biofilm development both in the animal host and in culture-based biofilm models that capture key aspects of in vivo signaling. In this work, we report the results of the transcriptomic profiling of two V. fischeri biofilm models followed by phenotypic validation and examination of novel signaling pathway architecture. Remarkable consistency between the models provides a strong basis for future studies using either approach or both. A subset of the factors identified by the approaches were validated in the work, and the body of transcriptomic data provides a number of leads for future studies in culture and during animal colonization.

Chlorination of antiviral drug ribavirin: Kinetics, nontargeted identification, and concomitant toxicity evolution.

Residual antiviral drugs in wastewater may increase the risk of generating transformation products (TPs) during wastewater treatment. Therefore, chlorination behavior and toxicity evolution are essential to understand the secondary ecological risk associated with their TPs. Herein, chlorination kinetics, transformation pathways, and secondary risks of ribavirin (RBV), one of the most commonly used broad-spectrum antivirals, were investigated. The pH-dependent second-order rate constants k increased from 0.18 M-1·s-1 (pH 5.8) to 1.53 M-1·s-1 (pH 8.0) due to neutral RBV and ClO- as dominant species. 12 TPs were identified using high-resolution mass spectrometry in a nontargeted approach, of which 6 TPs were reported for the first time, and their chlorination pathways were elucidated. The luminescence inhibition rate of Vibrio fischeri exposed to chlorinated RBV solution was positively correlated with initial free active chlorine, probably due to the accumulation of toxic TPs. Quantitative structure-activity relationship prediction identified 7 TPs with elevated toxicity, concentrating on developmental toxicity and bioconcentration factors, which explained the increased toxicity of chlorinated RBV. Overall, this study highlights the urgent need to minimize the discharge of toxic chlorinated TPs into aquatic environments and contributes to environmental risk control in future pandemics and regions with high consumption of antivirals.

Impact of modeled microgravity stress on innate immunity in a beneficial animal-microbe symbiosis.

The innate immune response is the first line of defense for all animals to not only detect invading microbes and toxins but also sense and interface with the environment. One such environment that can significantly affect innate immunity is spaceflight. In this study, we explored the impact of microgravity stress on key elements of the NFκB innate immune pathway. The symbiosis between the bobtail squid Euprymna scolopes and its beneficial symbiont Vibrio fischeri was used as a model system under a simulated microgravity environment. The expression of genes associated with the NFκB pathway was monitored over time as the symbiosis progressed. Results revealed that although the onset of the symbiosis was the major driver in the differential expression of NFκB signaling, the stress of simulated low-shear microgravity also caused a dysregulation of expression. Several genes were expressed at earlier time points suggesting that elements of the E. scolopes NFκB pathway are stress-inducible, whereas expression of other pathway components was delayed. The results provide new insights into the role of NFκB signaling in the squid-vibrio symbiosis, and how the stress of microgravity negatively impacts the host immune response. Together, these results provide a foundation to develop mitigation strategies to maintain host-microbe homeostasis during spaceflight.

Bacterial growth dynamics in a rhythmic symbiosis.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for ∼6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage.

Ecotoxicity assessment of ashes from calcium-rich fuel combustion: contrasting results and regulatory implications.

The European Union's (EU) regulation for the waste classification based on their ecotoxicological hazard (hazardous property HP14) came into force on 5 July 2018. The regulation advocates the utilisation of computational formulae for the hazard classification of waste associated with hazardous property HP14. Concurrently, ecotoxicological testing remains an alternative. To date, the absence of a consensus regarding test organisms and methodologies has vested EU member states with autonomy in determining the approach for conducting ecotoxicity assessments. The discussions on waste classification are also ongoing globally, namely the discussions under the Basel Convention. This paper endeavours to elucidate whether the widely employed test organisms, Daphnia magna and Aliivibrio fischeri, may serve as suitable indicators for the evaluation of the ecotoxicity of waste. The research is grounded in the examination of ashes derived from a combustion process of calcium-rich fuel. Ecotoxicity testing was conducted on 14 ash samples with a liquid-to-solid ratio of 10:1. The results of the Aliivibrio fischeri testing indicated that all 14 ash samples were non-hazardous in terms of their ecotoxicity. However, the results of the Daphnia magna testing showed the opposite, suggesting that the ash samples may have the potential to be ecotoxic. This study offers valuable insights into ecotoxicity assessment and waste classification, emphasising the need for scientific rigour and comprehensive understanding before making regulatory decisions. It also situates its findings within the broader global context of waste management discussions, particularly those related to international agreements like the Basel Convention.

Long-term toxicity assessment of antibiotics against Vibrio fischeri: Test method optimization and mixture toxicity prediction.

The current luminescent bacteria test for acute toxicity with short contact time was invalid for antibiotics, and the non-uniformed contact times reported in the literature for long-term toxicity assessment led to incomparable results. Herein, a representative long-term toxicity assessment method was established which unified the contact time of antibiotics and Vibrio fischeri within the bioluminescence increasing period (i.e. 10-100% maximum luminescence) of control samples. The effects of excitation and detoxification of antibiotics such as ß-lactams were discovered. Half maximal inhibitory concentration (IC50) of toxic antibiotics (0.00069-0.061 mmol/L) obtained by this method was 2-3 orders of magnitude lower than acute test, quantifying the underestimated toxicity. As antibiotics exist in natural water as mixtures, an equivalent concentration addition (ECA) model was built to predict mixture toxicity based on physical mechanism rather than mathematical method, which showed great fitting results (R2 = 0.94). Furthermore, interaction among antibiotics was investigated. Antibiotics acting during bacterial breeding period had strong synergistic inhibition (IC50 relative deviation from 0.1 to 0.6) such as macrolides and quinolones. Some antibiotics produced increasing synergistic inhibition during concentration accumulation, such as macrolides. The discharge of antibiotics with severe long-term toxicity and strong synergistic inhibition effect should be seriously restricted.