Single Strain Research Articles

Lactococcus garvieae as a Novel Pathogen in Cultured Pufferfish (Takifugu obscurus) in China

In October 2023, a disease outbreak in pufferfish (Takifugu obscurus) farms in Zhongshan City, Guangdong, China, caused high mortality. Diseased fish (mean length: 15 ± 1 cm) exhibited swimming disorders, fin rot, hemorrhage, and an enlarged spleen. Histopathological observations generally revealed inflammation, necrosis, and congestion in the spleen, kidneys, and brain tissues. The most severe pathological changes included interstitial edema and tubular atrophy in the kidneys, hemosiderin deposition in the spleen, massive red blood cell infiltration, and a decrease in lymphocytes. A single strain of bacteria (Tol-1) was isolated from the diseased pufferfish and identified as a Gram-positive streptococcus strain, exhibiting α-hemolysis on sheep blood agar plates. Through biochemical characterization, 16S rDNA sequencing, morphological analysis, and specific primer-based identification, the Tol-1 strain was identified as Lactococcus garvieae, serotype I. Antimicrobial susceptibility testing indicated that Tol-1 was sensitive to Chloramphenicol, Ampicillin, Cephalexin, and Doxycycline, but resistant to Kanamycin, Gentamicin and Ciprofloxacin. In addition, 15 common virulence factors were detected in the Tol-1 strain, including adhPav, adhPsaA, adhC I–II, adh, and hly 1–3. Pufferfish (mean length: 17 ± 1 cm) subjected to artificial infection via intraperitoneal injection (IP) with the Tol-1 strain exhibited clinical symptoms and histopathological damage similar to those observed in naturally infected fish. An infection dose of 1 × 105 CFU/fish resulted in 80% mortality. The study fulfilled Koch’s postulates, indicating that the disease outbreak in pufferfish was caused by L. garvieae, which exhibited a high mortality rate in pufferfish despite the subtle clinical symptoms. These results serve as a warning for pufferfish farming areas and provide a scientific basis for future prevention and control efforts.

Pan-genome-scale metabolic modeling of Bacillus subtilis reveals functionally distinct groups.

As the volume of genomic data and computational power have increased, so has the number of genome-scale metabolic models. These models encapsulate the totality of metabolic functions for a given organism. Bacillus subtilis strain 168 is one of the first bacteria for which a metabolic network was reconstructed. Since then, several updated reconstructions have been generated for this model microorganism. Here, we expand the metabolic model for a single strain into a pan-genome-scale model, which consists of individual models for 481 B. subtilis strains. By evaluating differences between these strains, we identified five distinct groups of strains, allowing for the rapid classification of any particular strain. Furthermore, this classification into five groups aids the rapid identification of suitable strains for any application.

Use of Direct‐Fed Microbes to Enhance Shrimp Resistance to a Vibrio Parahaemolyticus Strain Causing Early Morality Syndrome (EMS)

ABSTRACTEarly mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPND) is an epizootic bacterial infection of shrimp that has had significant negative impact on the global shrimp aquaculture industry. This disease is largely attributed to pathogenic Vibrio parahaemolyticus EMS strains (VP‐EMS). Application of probiotics was examined for control of pathogenic VP‐EMS infections in Pacific white shrimp (Litopenaeus vannamei) under laboratory‐scale conditions. Two probiotic products, a single strain (O14VRQ) of Bacillus subtilis and a blend (Plus10) of five strains of Bacillus from four different species (Bacillus amyloliquifaciens, Bacillus megaterium, Bacillus brevis, and two distinct strains of Bacillus subtilis), were evaluated at various concentrations as feed additives (applied as a top coat on commercial feed) or dosed directly into the culture water. Two trials were conducted in which shrimp were fed either a control feed (no probiotics) or probiotic‐coated feed for 7 days prior to be being challenged with VP‐EMS. Shrimp were observed for clinical signs of disease and mortalities during the disease challenge study. These experiments demonstrated that while both Bacillus probiotic products were shown to significantly (p < 0.05) improve shrimp survival, the O14VRQ strain provided the most consistent protection across the trials in top‐coated feed. Overall, better efficacy was observed with probiotic‐coated feeds. Findings were directly and positively correlated with concentration for top‐coated and even more so for direct in‐tank applications. Collectively, these findings contribute to our understanding about how probiotic strains could be applied to enhance shrimp health in aquaculture.

Comprehensive analysis of biotransformation pathways and products of chloramphenicol by Raoultella Ornithinolytica CT3: Pathway elucidation and toxicity assessment

Microbial degradation of chloramphenicol (CAP) has become important for reducing the adverse impact of environmental pollution with antibiotics. Although several pathways for CAP degradation have been identified in various bacteria, multiple metabolic pathways and their respective intermediate metabolites within a single strain are rarely reported. Here, Raoultella ornithinolytica CT3 was first isolated from silkworm excrement using CAP as the sole carbon source, and 100 mg/L CAP was almost completely degraded within 48 h. The biodegradation type of CAP followed first-order kinetics. Twenty-two CAP biotransformation products were identified using high-performance liquid chromatography and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. The CAP biotransformation pathways were predicted mainly in the acetylation and auxiliary pathways of propionylation and butyrylation. The toxicity of CAP biotransformation products was evaluated using the ecological structure-activity relationship (ECOSAR) model and biological indicators. The results showed that the toxicity of the intermediate metabolites changed slightly, but the final metabolite was harmless to the environment. Genomic analysis predicted that genes encoding acetyltransferase, amido-linkage hydrolase, nitroreductase, haloacetate dehalogenase, and protocatechuate 3,4-dioxygenase were associated with CAP biodegradation. This study provides new insights into the microbial degradation pathway of CAP and constitutes an ecological safety assessment for CAP-contaminated environments.

Identification of a cellular role of hemolysin co-regulatory protein (Hcp) in Vibrio alginolyticus modulating substrate metabolism and biofilm formation by cAMP-CRP

Cyclic AMP (cAMP) and cAMP receptor protein (CRP) system controls catabolic enzyme expression based on metabolite concentrations in bacteria. Hemolysin co-regulatory protein (Hcp) is well known as a molecular chaperone for virulence factor secretion of the type VI secretion system (T6SS). However, the intracellular role of Hcp involving in bacterial physiological processes remains unknown. To clarify that, we constructed a single hcp mutant strain and analyzed their effects on the physiological processes of Vibrio alginolyticus. The omics results revealed the extensive involvement of Hcp in the catabolic metabolism in bacteria. Simultaneously, Hcp1 and Hcp2 played opposing regulatory roles on the bacterial growth, biofilm formation, and intracellular cAMP-CRP levels during cultivation in a glucose medium. Furthermore, the interacting protein screening and co-immunoprecipitation (Co-IP) assays confirmed that the glucose-specific phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) enzyme IIA component (EIIAglc) was a key interacting partner with Hcp proteins as well as class I adenylyl cyclase (AC-I) in Vibrio alginolyticus. These results indicated that, to achieve cellular homeostasis, Hcp1 and Hcp2 might exert antagonistic and synergistic effects, respectively, on the interaction between EIIAglc and AC thus cooperatively regulating intracellular cAMP-CRP production.

Characterization of two phosphatase 2 C domain-containing proteins PPM2A and PPM2B in Toxoplasma gondii

Protein phosphatases Mg2+/Mn2+ dependent (PPMs), serine/threonine phosphatases, are widely distributed in apicomplexan parasites, and Toxoplasma gondii possesses the largest number of PPMs in the apicomplexan parasites. Though the function of some PPMs has been characterized in T. gondii, much less is known about two phosphatase 2C domain-containing proteins, PPM2A and PPM2B. PPM2A was identified as one of Toxoplasma Calmodulin's interacting proteins through proximity-based protein interaction BioID technology in the previous study, and PPM2B was the homolog of PPM2A in T. gondii. In this study, PPM2A was distributed in the whole tachyzoite of T. gondii, and PPM2B was mainly distributed in the cytoplasm by inserting a 10HA tag in the C-terminus of the two genes in the RH∆ku80 strain. PPM2A knockout (Δppm2a), PPM2B knockout (Δppm2b), and double knockout (ΔΔ) in RHΔhxgprt type I strain under CRISPR-Cas9 system did not result in intracellular replication defect. Besides, mouse experiments demonstrated that PPM2A, PPM2B, and double knockout did not reduce the pathogenicity of T. gondii compared with the RH∆hxgprt strain. However, the plaque size of these single knockout and double knockout strains were smaller than that in the control RH∆hxgprt strain. Our results provide new insight into the function of PPMs in the pathogenesis of T. gondii.

Diversity and Complexity of CTXΦ and Pre-CTXΦ Families in Vibrio cholerae from Seventh Pandemic.

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTXETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic.

Population distribution characteristics of mating type genes and genetic stability in Morchella sextelata.

The reproductive mode of morels (Morchella spp.) is governed by mating type genes, specifically MAT1-1 and MAT1-2. This study investigated the presence of mating type genes at various growth stages and in different parts of cultivated Morchella sextelata. This study revealed significant fluctuations in the detection ratio of the two mating type genes during ascocarps growth. Single ascospore strains with MAT1-1, MAT1-2 and both mating types were selected for experimentations. Stress stimuli including H2O2, Congo red and NaCl were introduced into the medium. Differences in the cultural and physiological characteristics of single spore strains were analyzed, and mating type genes were identified after subculturing to assess their stability. The results indicated that a total of 297 samples with a single mating type gene were detected in 480 samples selected from the five stages of fruiting body growth, accounting for 61.9%. Stress exposure influenced colony morphology, mycelial growth rate, and biomass, leading to significant increases in malondialdehyde content and osmotic adjustment compounds, including soluble protein and proline. Physiological and biochemical parameters varied among the three mating type strains under different stress conditions. Principal component analysis was used to calculate the weight values, which showed that the MAT1-2 strain exhibited the highest tolerance to chemical stresses, particularly oxidative stress. Subculturing under stress revealed that single mating type strains ceased growth by the 8th generation, whereas both mating type strains could continue to the 15th generation without loss of mating type genes, indicating broader environmental adaptability and higher viability. These findings offer novel insights into mating type gene function and serve as a scientific foundation for the development of high-yield, stress-resistant morel varieties.

Engineering Microbial Consortia as Living Materials: Advances and Prospectives.

The field of Engineered Living Materials (ELMs) integrates engineered living organisms into natural biomaterials to achieve diverse objectives. Multiorganism consortia, prevalent in both naturally occurring and synthetic microbial cultures, exhibit complex functionalities and interrelationships, extending the scope of what can be achieved with individual engineered bacterial strains. However, the ELMs comprising microbial consortia are still in the developmental stage. In this Review, we introduce two strategies for designing ELMs constituted of microbial consortia: a top-down strategy, which involves characterizing microbial interactions and mimicking and reconstructing natural ecosystems, and a bottom-up strategy, which entails the rational design of synthetic consortia and their assembly with material substrates to achieve user-defined functions. Next, we summarize technologies from synthetic biology that facilitate the efficient engineering of microbial consortia for performing tasks more complex than those that can be done with single bacterial strains. Finally, we discuss essential challenges and future perspectives for microbial consortia-based ELMs.

Three separate pathways in Rhizobium leguminosarum maintain phosphatidylcholine biosynthesis, which is required for symbiotic nitrogen fixation with clover.

Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.

Endophytic consortium exhibits varying effects in mitigating cadmium toxicity in rice cultivars with distinct cadmium accumulation capacities

Cadmium (Cd)-contaminated rice is the main source of Cd exposure that threatens food safety and human health. While plant growth-promoting bacteria (PGPB) have been reported to reduce Cd toxicity in rice, the effect of endophytic consortium is less understood compared to the single strain. Here, we reported that the PGPB consortium consisting of Cd-tolerant endophytic bacteria Pseudomonas sp. 4N2 and Bacillus sp. TB1 increased extracellular polymeric substance secretion and demonstrated a higher Cd removal rate compared to individual 4N2 and TB1. Further, two rice cultivar, namely the low-Cd-accumulating (LA) cultivar 728B and the high-Cd-accumulating (HA) cultivar BB, were inoculated with the 4N2-TB1 consortium. As expected, the consortium had a more pronounced effect on 728B, from which the endophytic bacteria were screened. The 4N2-TB1 consortium was found to facilitate the growth of rice seedlings and enhance their antioxidation activity. Moreover, the consortium significantly reduced Cd transfer coefficient from root to shoot through Cd immobilization in rice roots, resulting in a reduction from 30 % to 6 % in 728B and from 31 % to 13 % in BB. Furthermore, alpha-diversity analysis indicated an increased diversity and richness of the root bacterial community after 4N2-TB1 inoculation. Also, redundancy analysis confirmed a positive correlation between rice biomass and Cd content with a specific assemblage of bacteria including Bacillus and Leifsonia. These results demonstrated that the 4N2-TB1 consortium may alleviate Cd stress in rice cultivars through recruiting PGPB and Cd-binding bacteria on the root surface, thus strengthening the immobilization of Cd in rice roots.

Antioxidant Peptides Derived from Cheese Products via Single and Mixed Lactobacillus Strain Fermentation.

Probiotics are used in cheese fermentation to endow the product with unique functional properties, such as enhanced flavor and aroma development through proteolysis and lipolysis. In this study, two probiotic Lactobacillus strains, Lactobacillus plantarum A3 and Lactobacillus reuteri WQY-1, were selected to develop new probiotic cheeses in the form of single- and mixed-strain starters. The results demonstrated that the L. plantarum A3 single-strain group and the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group exhibited superior product performance, particularly the release of functional hydrolysates during cheese ripening. Furthermore, Label-free quantitative proteomic analysis revealed 26 unique antioxidant peptides in the L. plantarum A3 single-strain group and 53 in the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group. Among these, CMENSAEPEQSLACQCL (β-lactoglobulin), CMENSAEPEQSLVCQCL (β-lactoglobulin), and IQYVLSR (κ-casein) have been found to possess potential antioxidant properties both in vitro and in vivo. This confirmed that milk-derived protein peptides in cheese products exhibit potential antioxidant functions through the hydrolysis of probiotic strains.

Solid-state fermentation with Bacillus subtillis co-cultured with probiotic Lactobacillus spp. enhances the bioactive peptides, nutritional and antioxidative potentials of tamarind seed

The effect of natural or controlled solid-state fermentation using single or mixed probiotic strains on the nutritional, bioactive and phytochemical constituents, and antioxidant activities of tamarind seeds was evaluated. Tamarind seeds were subjected to either controlled solid-state fermentation (37 °C, 72 h) using either Bacillus subtilis only or Bacillus subtilis co-cultured with probiotics Lactobacillus spp. (Lactobacillus fermentum, Lactobacillus rhamnosus GG, Lactobacillus brevis, and Lactobacillus delbrueckii) or natural fermentation while unfermented tamarind seeds served as control. The changes in the nutritional composition, peptide content, antioxidative properties (Ferric reducing antioxidant power (FRAP) and DPPH radical scavenging activities) and total viable count of probiotics in fermented tamarind seed were evaluated using standard methods. The peptide content, crude protein, fat, and fibre (100.19 mg/g, 51.35, 9.50 and 5.10%), and FRAP and DPPH (0.55 and 21.60%) in unfermented tamarind seed were significantly (p < 0.05) lower than fermented tamarind seed (271.00–308 mg/g, 12, 62.84–64.02, 12.21–13.11 and 6.01–6.90, and 1.42–2.92 and 26.66–35.70%, respectively), after 72 h of fermentation. The total viable count in the tamarind seed fermented with Bacillus subtilis only was 1.2 × 109 CFU/g while that co-cultured with probiotics ranged from 6 × 108 to 9.9 × 108 CFU/g after 72 h of fermentation. This study showed that a controlled solid-state fermentation using probiotics Lactobacillus co-cultured with Bacillus subtilis can significantly increase the peptide contents, and enhance the nutritional and antioxidant activities of tamarind seed while producing a condiment with significant probiotic potential.

The bacterial and yeast microbiota in livestock forages in Hungary

BackgroundAlong bacteria, yeasts are common in forages and forage fermentations as spoilage microbes or as additives, yet few studies exist with species-level data on these fungi’s occurrence in feedstuff. Active dry yeast and other yeast-based products are also common feed additives in animal husbandry. Here, we aimed to characterize both fermented and non-fermented milking cow feedstuff samples from Hungary to assess their microbial diversity in the first such study from Central Europe.ResultsWe applied long-read bacterial metabarcoding to 10 fermented and 25 non-fermented types of samples to assess bacterial communities and their characteristics, surveyed culturable mold and yeast abundance, and identified culturable yeast species. Fermented forages showed the abundance of Aerococcaceae, Bacillaceae, Brucellaceae, Lactobacillaceae, Staphylococcaceae, and Thermoactinomycetaceae, non-fermented ones had Cyanothecaceae, Enterobacteriaceae, Erwiniaceae, Gomontiellaceae, Oxalobacteraceae, Rhodobiaceae, Rickettsiaceae, and Staphylococcaceae. Abundances of bacterial families showed mostly weak correlation with yeast CFU numbers, only Microcoleaceae (positive) and Enterococcaceae and Alcaligenaceae (negative correlation) showed moderate correlation. We identified 14 yeast species, most commonly Diutina rugosa, Pichia fermentans, P. kudriavzevii, and Wickerhahomyces anomalus. We recorded S. cerevisiae isolates only from animal feed mixes with added active dry yeast, while the species was completely absent from fermented forages. The S. cerevisiae isolates showed high genetic uniformity.ConclusionOur results show that both fermented and non-fermented forages harbor diverse bacterial microbiota, with higher alpha diversity in the latter. The bacterial microbiome had an overall weak correlation with yeast abundance, but yeasts were present in the majority of the samples, including four new records for forages as a habitat for yeasts. Yeasts in forages mostly represented common species including opportunistic pathogens, along with a single strain of Saccharomyces used as a feed mix additive.

Biodegradation of low-density polyethylene by mixed fungi composed of Alternaria sp. and Trametes sp. isolated from landfill sites

With the development of industry and modern manufacturing, nondegradable low-density polyethylene (LDPE) has been widely used, posing a rising environmental hazard to natural ecosystems and public health. In this study, we isolated a series of LDPE-degrading fungi from landfill sites and carried out LDPE degradation experiments by combining highly efficient degrading fungi in pairs. The results showed that the mixed microorganisms composed of Alternaria sp. CPEF-1 and Trametes sp. PE2F-4 (H-3 group) had a greater degradation effect on heat-treated LDPE (T-LDPE). After 30 days of inoculation with combination strain H-3, the weight loss rate of the T-LDPE film was approximately 154% higher than that of the untreated LDPE (U-LDPE) film, and the weight loss rate reached 0.66 ± 0.06%. Environmental scanning electron microscopy (ESEM) and Fourier transform infrared spectroscopy (FTIR) were used to further investigate the biodegradation impacts of T-LDPE, including the changes on the surface and depolymerization of the LDPE films during the fungal degradation process. Our findings revealed that the combined fungal treatment is more effective at degrading T-LDPE than the single strain treatment, and it is expected that properly altering the composition of the microbial community can help lessen the detrimental impact of plastics on the environment.

Metabolic engineering of Escherichia coli for high-level production of benzyl acetate from glucose

BackgroundBenzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate.ResultsTwo biosynthetic routes based on the CoA-dependent β-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent β-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation.ConclusionsWe were able to establish the whole pathway for benzyl acetate based on the CoA-dependent β-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.Graphical abstract

Biodegradation of DDT using multi-species mixtures: Fromgenome-mining prediction to practical assessment.

DDT (dichlorodiphenyltrichloroethane) is a commonly used insecticide that is recalcitrant and highly stable in the environment. Currently, DDT residue contamination, especially in agricultural soil, is still a concern in many countries, threatening human health and the environment. Among the approaches to resolve such an issue, novel biodegradation-based methods are now preferred to physicochemical methods, due to the sustainability and the effectiveness of the former. In this study, we explored the possibility of building mixed microbial cultures that can offer improved DDT-degrading efficiencies and be more environmentally transilient, based on genome annotation using the KEGG database and prediction of interactions between single strains using the obtained metabolic maps. We then proposed 10 potential DDT-degrading mixed cultures of different strain combinations and evaluated their DDT degradation performances in liquid, semi-solid and solid media. The results demonstrated the superiority of the mixtures over the single strains in terms of degrading DDT, particularly in a semi-solid medium, with up to 40-50% more efficiency. Not only did the mixed cultures degrade DDT more efficiently, but they also adapted to broader spectra of environmental conditions. The three best DDT-degrading and transilient mixtures were selected, and it turned out that their component strains seemed to have more metabolic interactions than those in the other mixtures. Thus, our study demonstrates the effectiveness of exploiting genome-mining techniques and the use of constructed mixed cultures in improving biodegradation.

Simulating the clinical manifestations and disease progression of human sepsis: A monobacterial injection approach for animal modeling

ABSTRACT Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, with great clinical heterogeneity, high morbidity, and high mortality. At the same time, there are many kinds of infection sources, the pathophysiology is very complex, and the pathogenesis has not been fully elucidated. An ideal animal model of sepsis can accurately simulate clinical sepsis and promote the development of sepsis-related pathogenesis, treatment methods, and prognosis. The existing sepsis model still uses the previous Sepsis 2.0 modelling standard, which has some problems, such as many kinds of infection sources, poor repeatability, inability to take into account single-factor studies, and large differences from clinical sepsis patients. To solve these problems, this study established a new animal model of sepsis. The model uses intravenous tail injection of a single bacterial strain, simplifying the complexity of multibacterial infection, and effectively solving the above problems.

The role of aroA and ppk1 in Aeromonas veronii pathogenicity and the efficacy evaluation of mutant strain AV-ΔaroA/ppk1 as a live attenuated vaccine

Aeromonas veronii is an opportunistic pathogen that poses great threat to aquaculture and human health, so there is an urgent need for green and efficient methods to deal with its infection. In this study, single and double gene deletion strains (AV-ΔaroA, AV-Δppk1 and AV-ΔaroA/ppk1) that can be stably inherited were constructed. Pathogenicity test showed that the toxicity of AV-ΔaroA and AV-ΔaroA/ppk1 was significantly lower compared to wild-type A. veronii. Biological characterization analysis revealed that the decrease in pathogenicity might be due to the declined growth, motility, biofilm formation abilities and the expression of virulence-related genes in mutants. Subsequently, we evaluated the efficacy of AV-ΔaroA/ppk1 as a live attenuated vaccine (LAV). Safety assessment experiments showed that AV-ΔaroA/ppk1 injected at a concentration of 3 × 107 CFU/mL was safe for C. carassius. The relative percentage survival of AV-ΔaroA/ppk1 was 67.85 %, significantly higher than that of the inactivated A. veronii, which had an RPS of 54.84 %. This improved protective effect was mainly attributed to the increased levels of A. veronii specific IgM antibody, enhanced alkaline phosphatase, lysozyme and superoxide dismutase activities, as well as higher expression levels of several immune related genes. Together, these findings deepen our understanding of the functional roles of aroA and ppk1 in A. veronii pathogenicity, provide a good candidate of LAV for A. veronii.

Single-Molecule Fingerprinting Reveals Different Growth Mechanisms in Seed Amplification Assays for Different Polymorphs of α-Synuclein Fibrils.

α-Synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson's disease (PD), have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson's disease and turns the presence of αSyn aggregates into a biomarker of the disease. It has become evident that αSyn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays. Here, we compared the properties of two well-described αSyn polymorphs. Using single-molecule techniques, we observed that one of the polymorphs had an increased tendency to undergo secondary nucleation and we showed that this could explain the differences in reactivity observed in in vitro seed amplification assay and cellular assays. Simulations and high-resolution microscopy suggest that a 100-fold difference in the apparent rate of growth can be generated by a surprisingly low number of secondary nucleation "points" (1 every 2000 monomers added by elongation). When both strains are present in the same seeded reaction, secondary nucleation displaces proportions dramatically and causes a single strain to dominate the reaction as the major end product.