Zebrafish Infection Research Articles

Anti-bacterial and anti-inflammatory properties of Vernonia arborea accelerate the healing of infected wounds in adult Zebrafish.

Management of wounds and healing under impaired conditions are the major challenges faced globally by healthcare workers. Phytocompounds which are anti-microbial and capable of modulating inflammation contribute to overall wound healing and regain of the lost structure and function especially in wounds impaired with polymicrobial infection. An acute cutaneous impaired wound model using adult zebrafish was validated to simulate mammalian wound pathophysiology. This model was used to evaluate phytofractions of Vernonia arborea in the present study, for reduction of infection; myeloperoxidase (MPO) as a marker of infection; neutrophil infiltration and resolution; kinetics of inflammatory cytokines; and wound repair kinetics (viz., nitrite levels and iNoS expression; reepithelisation). Four fractions which were active in-vitro against five selected wound microbes were shown to reduce ex-vivo microbial bioburden upto 96% in the infected wound tissue. The reduction in CFU correlated with the neutrophil kinetics and MPO enzyme levels in the treated, wound infected zebrafish. Expression of pro-inflammatory cytokines (IL-6 and TNF-α) was downregulated while upregulating anti-inflammatory cytokine (IL-10), and nitric oxide signalling with fourfold increase in iNOS expression. The adult zebrafish wound model could well serve as a standard tool for assessing phytoextracts such as V. arborea for wound healing with anti-microbial properties.

Gut dysbiosis induced by florfenicol increases susceptibility to Aeromonas hydrophila infection in Zebrafish Danio rerio after the recommended withdrawal period.

Florfenicol (FFC) is a broad-spectrum antibiotic approved by the U.S. Food and Drug Administration to treat both systemic and external bacterial infections in food fish. The objective of this study was to evaluate the effect of FFC-medicated feed on the gut microbiota of Zebrafish Danio danio to determine (1) if the therapeutic dose of FFC-medicated feed induces dysbiosis and (2) if fish with altered gut microbiota were more susceptible to subsequent infection by the common opportunistic fish pathogen Aeromonas hydrophila. Zebrafish that were treated with regular and FFC-medicated feeds were artificially challenged with A. hydrophila at the end of the recommended 15-day antibiotic withdrawal period. The gut microbiota of the Zebrafish at different stages was analyzed using 16S ribosomal RNA gene sequencing. Our results found that FFC-medicated feed induced disruption of the gut microbiota. Dysbiosis was observed in all treated groups, with a significant increase in bacterial diversity, and was characterized by a remarkable bloom of Proteobacteria and a drastic decline of Mycoplasma and Cetobacterium in treated animals but without noticeable clinical signs or mortalities. In addition, the increase of Proteobacteria was not significantly reduced after the recommended 15-day withdrawal period, and the Zebrafish treated with FFC-medicated feed exhibited a significantly higher mortality rate when they were subsequently challenged with A. hydrophila compared to the control (regular feed) groups. Interestingly, the most dramatic changes in the gut microbiome composition occurred at the transition time between the late stage of the medicated treatment and the beginning of the withdrawal period instead of the time during the Aeromonas infection. The administration of FFC-medicated feed at the recommended dose induced gut dysbiosis in Zebrafish, and fish did not recover to the baseline after the recommended withdrawal period. Our findings suggest that the use of antibiotics in fish elicits a response similar to those previously described in mammals and possibly makes the host more susceptible to subsequent infections of opportunistic pathogens. This study using a controlled model system suggests that antibiotics in aquaculture may have long-term effects on the general well-being of the fish.

Dynamics of macrophage polarization support Salmonella persistence in a whole living organism.

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

Viperin mutation is linked to immunity, immune cell dynamics, and metabolic alteration during VHSV infection in zebrafish.

Viperin is a prominent antiviral protein found in animals. The primary function of Viperin is the production of 3'-deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP), an inhibitory nucleotide involved in viral RNA synthesis. Studies in mammalian models have suggested that ddhCTP interferes with metabolic proteins. However, this hypothesis has yet to be tested in teleost. In this study, the role of Viperin in regulating metabolic alterations during viral hemorrhagic septicemia virus (VHSV) infection was tested. When infected with VHSV, viperin -/- fish showed considerably higher mortality rates. VHSV copy number and the expression of the NP gene were significantly increased in viperin -/- fish. Metabolic gene analysis revealed significant differences in soda, hif1a, fasn, and acc expression, indicating their impact on metabolism. Cholesterol analysis in zebrafish larvae during VHSV infection showed significant upregulation of cholesterol production without Viperin. In vitro analysis of ZF4 cells suggested a considerable reduction in lipid production and a significant upregulation of reactive oxygen species (ROS) generation with the overexpression of viperin. Neutrophil and macrophage recruitment were significantly modulated in viperin -/- fish compared to the wild-type (WT) fish. Thus, we have demonstrated that Viperin plays a role in interfering with metabolic alterations during VHSV infection.

The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model.

Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

Influence of colour background on anti-viral activity against viral haemorrhagic septicaemia virus in zebrafish regulated by circadian rhythm signalling pathway.

The influence of colour background on the regulation of behavioural and physiological responses in zebrafish is widely recognized. However, its specific effect on virus infection in zebrafish remains unclear. This study aimed to explore the susceptibility of zebrafish to viral haemorrhagic septicaemia virus (VHSV) infection in relation to background colour, investigate the underlying mechanisms, and elucidate the involvement of key molecules, using proteomic and gene expression analyses. The results revealed that zebrafish housed in a blue tank exhibited higher survival rates and considerably reduced VHSV replication compared to those housed in a yellow tank. Further, up-regulation of apolipoprotein 1 (APOA1) was identified as a crucial shared mechanism associated with survival in zebrafish exposed to VHSV infection and reared in a blue background. The mRNA expression level of bmal1a, a core gene involved in the circadian rhythm, was consistently downregulated in fish from the blue tank compared to fish from the yellow tank, regardless of infection status. Subsequently, zebrafish in the blue tank were exposed to daylight conditions to stimulate per2 and pgc1a expression, aiming to investigate their potential impact on VHSV infection. The validity of these interconnected events, triggered by background colour, involving APOA1 up-regulation, circadian rhythm modulation, and antiviral responses, was confirmed by treatments with hesperetin and cyclosporine A, an activator and inhibitor of apoa1 respectively. Our findings revealed the influence of background colour on the apoa1 expression level, thus establishing the involvement of a novel network through circadian rhythm signalling.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.

Chitosan nanoparticles-based in vivo delivery of miR-155 modulates the Viral haemorrhagic septicaemia virus-induced antiviral immune responses in zebrafish (Danio rerio)

Viral haemorrhagic septicaemia virus (VHSV) is one of the highly pathogenic virus, which causes viral haemorrhagic septicaemia disease in both marine and freshwater fish. Micro RNA-155 (miRNA-155) is a multifunctional small non-coding RNA and it involves regulation of immune responses during viral infection. In this study, dre-miR-155 mimics were encapsulated into chitosan nanoparticles (CNPs). Resulted encapsulated product (miR-155-CNPs) was investigated for its immunomodulation role in zebrafish during experimentally challenged VHSV infection. Successful encapsulation of dre-miR-155 mimics into CNPs was confirmed through average nanoparticle (NPs) size (341.45 ± 10.00 nm), increased encapsulation efficiency percentage (98.80%), bound dre-miR-155 with chitosan, sustained release in vitro (up to 40%), and the integrity of RNA. Overexpressed miR-155 was observed in gills, muscle, and kidney tissues (5.42, 19.62, and 140.72-folds, respectively) after intraperitoneal delivery of miR-155-CNPs into zebrafish upon VHSV infection (miR-155-CNPs + VHSV). The miR-155-CNPs + VHSV infected fish had the highest cumulative survival (85%), which was associated with low viral copy numbers. The miR-155-overexpressing fish showed significantly decreased expression of ifnγ, irf2bpl, irf9, socs1a, il10, and caspase3, compared to that of the miR-155 inhibitor + VHSV infected fish group. In contrast, il1β, tnfα, il6, cd8a, and p53 expressions were upregulated in miR-155-overexpressed zebrafish compared to that of the control. The overall findings indicate the successful delivery of dre-miR-155 through miR-155-CNPs that enabled restriction of VHSV infection in zebrafish presumably by modulating immune gene expression.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Sequential infection of human norovirus and Salmonella enterica resulted in higher mortality and ACOD1/IRG1 upregulation in zebrafish larvae

Human norovirus (HNoVs) and Salmonella are both very important foodborne pathogens with mixed infection of HNoV and Salmonella reported clinically. With the use of model organism zebrafish (Danio rerio), it was observed that the sequential infection of HNoVs and Salmonella caused lower survival rates (12.5 ± 4.2%) than the single-pathogen infection by Salmonella (31.6 ± 7.3%, P < 0.05) or HNoVs (no mortality observed). Gene expression study with the use of RT-PCR and global transcriptomic analysis revealed that the mortality of zebrafish larvae was very likely due to the harmful inflammatory responses. Specifically, it was noted that the genes encoding aconitate decarboxylase 1 (ACOD1), also known as immunoresponsive gene 1 (IRG1), were significantly upregulated in the sequentially infected zebrafish larvae. The expression of acod1 could lead to mitochondrial reactive oxygen species (ROS) production. The ROS levels were indeed higher in sequentially infected zebrafish larvae than the single-pathogen infected ones (P < 0.05). An immersion treatment of glutathione or citraconate did not affect the microbial loads of HNoVs and Salmonella but significantly reduced the ROS levels and protected the zebrafish larvae by inducing higher survival rates in the sequentially infected zebrafish larvae (P < 0.05). Taken together, this study accumulated new knowledge over the function of ACOD1/IRG1 pathway in infectious diseases, and proposed possible treatment strategies accordingly.

Development of phage resistance in multidrug-resistant Klebsiella pneumoniae is associated with reduced virulence: a case report of a personalised phage therapy

ObjectivesPhage-resistant bacteria often emerge rapidly when performing phage therapy. However, the relationship between the emergence of phage-resistant bacteria and improvements in clinical symptoms is still poorly understood. MethodsAn inpatient developed a pulmonary infection caused by multidrug-resistant Klebsiella pneumoniae. He received a first course of treatment with a single nebulized phage (ΦKp_GWPB35) targeted at his bacterial isolate of Kp7450. After 14 days, he received a second course of treatment with a phage cocktail (ΦKp_GWPB35+ΦKp_GWPA139). Antibiotic treatment was continued throughout the course of phage therapy. Whole-genome analysis was used to identify mutations in phage-resistant strains. Mutated genes associated with resistance were further analysed by generating knockouts of Kp7450 and by measuring phage adsorption rates of bacteria treated with proteinase K and periodate. Bacterial virulence was evaluated in mouse and zebrafish infection models. ResultsPhage-resistant Klebsiella pneumoniae strains emerged after the second phage treatment. Comparative genomic analyses revealed that fabF was deleted in phage-resistant strains. The fabF knockout strain (Kp7450ΔfabF) resulted in an altered structure of lipopolysaccharide (LPS), which was identified as the host receptor for the therapeutic phages. Virulence evaluations in mice and zebrafish models showed that LPS was the main determinant of virulence in Kp7450 and alteration of LPS structure in Kp7450ΔfabF, and the bacteriophage-resistant strains reduced their virulence at cost. DiscussionThis study may shed light on the mechanism by which some patients experience clinical improvement in their symptoms post phage therapy, despite the incomplete elimination of pathogenic bacteria.

Ablation of myd88 alters the immune gene expression and immune cell recruitment during VHSV infection in zebrafish

Myeloid differentiation primary response protein-88 (MYD88) is an essential adaptor molecule in pathogen-related pattern recognition signaling pathways. Toll-like and interleukin receptors recognize numerous signals and are funneled through MyD88 to express genes responsible for the innate and adaptive immune systems. In the present study, the relevance of MyD88 in viral hemorrhagic septicemia virus (VHSV) was investigated by generating myd88−/− zebrafish. The model was challenged with VHSV, and viral propagation was quantified by evaluating clinical symptoms, mortality, and VHSV copy number. The infected fish showed abnormal morphologies, such as subcutaneous hemorrhages, abdominal swelling, and bulging eyes, which were comparatively more intense in myd88−/− fish than in the wild-type. An injury infection experiment conducted in zebrafish larvae indicated a substantial spread of VHSV in the wound site. The number of neutrophils and macrophages recruited to the wounded area were markedly reduced in myd88−/− fish. According to gene expression analysis, VHSV NP gene expression was considerably upregulated in myd88−/− fish. Substantial gene expression and immune cell marker modulation were observed in the mutant model compared to that in the wild-type. These results suggest that the lack of a significant adaptor protein for immune signal transduction results in enhanced VHSV replication.

Antibacterial activity of the novel oxazolidinone contezolid (MRX-I) against Mycobacterium abscessus.

To evaluate contezolid (MRX-I) antibacterial activity against Mycobacterium abscessus in vitro and in vivo and to assess whether MRX-I treatment can prolong survival of infected zebrafish. MRX-I inhibitory activity against M. abscessus in vitro was assessed by injecting MRX-I into zebrafish infected with green fluorescent protein-labelled M. abscessus. Thereafter, infected zebrafish were treated with azithromycin (AZM), linezolid (LZD) or MRX-I then maximum tolerated concentrations (MTCs) of drugs were determined based on M. abscessus growth inhibition using one-way ANOVA. Linear trend analysis of CFU counts and fluorescence intensities (mean ± SE values) was performed to detect linear relationships between MRX-I, AZM and LZD concentrations and these parameters. MRX-I anti-M. abscessus minimum inhibitory concentration (MIC) and MTC were 16 μg/mL and 15.6 μg/mL, respectively. MRX-I MTC-treated zebrafish fluorescence intensities were significantly lower than respective LZD group intensities (whole-body: 439040 ± 3647 vs. 509184 ± 23064, p < 0.01); head: 74147 ± 2175 vs. 95996 ± 8054, p < 0.05). As MRX-I concentration was increased from 0.488 μg/mL to 15.6 μg/mL, zebrafish whole-body, head and heart fluorescence intensities decreased. Statistically insignificant differences between the MRX-I MTC group survival rate (78.33%) vs. corresponding rates of the 62.5 μg/mL-treated AZM MTC group (88.33%, p > 0.05) and the 15.6 μg/mL-treated LZD MTC group (76.67%, p > 0.05) were observed. MRX-I effectively inhibited M. abscessus growth and prolonged zebrafish survival when administered to M. abscessus-infected zebrafish, thus demonstrating that MRX-I holds promise as a clinical treatment for human M. abscessus infections.

Shigella Serotypes Associated With Carriage in Humans Establish Persistent Infection in Zebrafish.

Shigella represents a paraphyletic group of enteroinvasive Escherichia coli. More than 40 Shigella serotypes have been reported. However, most cases within the men who have sex with men (MSM) community are attributed to 3 serotypes: Shigella sonnei unique serotype and Shigella flexneri 2a and 3a serotypes. Using the zebrafish model, we demonstrate that Shigella can establish persistent infection in vivo. Bacteria are not cleared by the immune system and become antibiotic tolerant. Establishment of persistent infection depends on the O-antigen, a key constituent of the bacterial surface and a serotype determinant. Representative isolates associated with MSM transmission persist in zebrafish, while representative isolates of a serotype not associated with MSM transmission do not. Isolates of a Shigella serotype establishing persistent infections elicited significantly less macrophage death in vivo than isolates of a serotype unable to persist. We conclude that zebrafish are a valuable platform to illuminate factors underlying establishment of Shigella persistent infection in humans.

Characterization and biocontrol efficacy of lytic phage (KPP-1) that infects multidrug resistant Klebsiella variicola.

Klebsiella variicola strain was identified from a natural water stream. Novel phage (KPP-1) infecting K. variicola was isolated and characterized. The biocontrol efficacy of KPP-1 against K. variicola-infected adult zebrafish was also investigated. The host K. variicola strain was resistant to six of the antibiotics tested and comprised the virulence genes kfuBC, fim, ureA, and Wza-Wzb-Wzccps. Morphological analysis by transmission electron microscopy revealed that KPP-1 has icosahedron head and tail structures. The latent period and burst size of KPP-1 were 20min and 88 PFU per infected cell, respectively, at a multiplicity of infection of 0.1. KPP-1 was stable over a broad pH range (3-11), temperature (4-50°C), and salinity (0.1-3%). KPP-1 inhibits the growth of K. variicola in vitro and in vivo. In the zebrafish infection model, treatment with KPP-1-infected K. variicola demonstrated 56% of cumulative survival. This suggests the possibility of developing KPP-1 as a potential biocontrol agent against multidrug-resistant K. variicola that belongs to the K. pneumoniae complex.

Oridonin Inhibits Mycobacterium marinum Infection-Induced Oxidative Stress In Vitro and In Vivo.

Prior to the COVID-19 pandemic, tuberculosis (TB) was the leading cause of death globally attributable to a single infectious agent, ranking higher than HIV/AIDS. Consequently, TB remains an urgent public health crisis worldwide. Oridonin (7a,20-Epoxy-1a,6b,7,14-tetrahydroxy-Kaur-16-en-15-one Isodonol, C20H28O6, Ori), derived from the Rabdosia Rrubescens plant, is a natural compound that exhibits antioxidant, anti-inflammatory, and antibacterial properties. Our objective was to investigate whether Ori's antioxidant and antibacterial effects could be effective against the infection Mycobacterium marinum (Mm)-infected cells and zebrafish. We observed that Ori treatment significantly impeded Mm infection in lung epithelial cells, while also suppressing inflammatory response and oxidative stress in Mm-infected macrophages. Further investigation revealed that Ori supplementation inhibited the proliferation of Mm in zebrafish, as well as reducing oxidative stress levels in infected zebrafish. Additionally, Ori promoted the expression of NRF2/HO-1/NQO-1 and activated the AKT/AMPK-α1/GSK-3β signaling pathway, which are both associated with anti-inflammatory and antioxidant effects. In summary, our results demonstrate that Ori exerts inhibitory effects on Mm infection and proliferation in cells and zebrafish, respectively. Additionally, Ori regulates oxidative stress by modulating the NRF2/HO-1/NQO-1 and AKT/AMPK-α1/GSK-3β signaling pathways.

WS07.02 Zebrafish infection model to assess persistence of Pseudomonas aeruginosa clinical isolates and treatment efficiency

Objectives: Pseudomonas aeruginosa is a main cause of chronic infections in patients with cystic fibrosis (CF). Our aim was to set up P. aeruginosa persistent infection in zebrafish (Danio rerio), a non-mammalian vertebrate model suitable for real-time imaging of infection and assessment of treatment efficacy.

Acquisition of a large virulence plasmid (pINV) promoted temperature-dependent virulence and global dispersal of O96:H19 enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired. IMPORTANCE Enteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America.

Bacterial clearance and anti-inflammatory effect of Withaferin A against human pathogen of Staphylococcus aureus in infected zebrafish

The emergence of antibiotic resistance is the most challenging factor for developing a proper drug to treat S. aureus infection. These bacterial pathogens can survive in fresh water and spread to various environments. Plant sources, especially pure compounds, are the material of interest amongst researchers for developing drugs of therapeutic value. Here, we report the bacterial clearance and anti-inflammatory potential of the plant compound Withaferin A, using the zebrafish infection model. The minimum inhibitory concentration of the Withaferin A was calculated as 80 µM against S. aureus. The DAPI/PI staining and scanning electron microscopy analysis showed the pore-forming mechanism of Withaferin A on the bacterial membrane. Along with the antibacterial activity, the results from the tube adherence test reveal the antibiofilm property of Withaferin A. In vivo studies were demonstrated to determine the effect of Withaferin A on survival, inflammatory response and behavioural changes during S. aureus infection. Staining zebrafish larvae with neutral red and Sudan black indicates a substantial decrease in the number of localized macrophages and neutrophils. The gene expression analysis showed the downregulation of inflammatory marker genes. Additionally, we observed the improvement in locomotory behaviour among Withaferin A treatment adult zebrafish. In conclusion, S. aureus can infect zebrafish and induces toxicological effect. In comparison, the results from in vitro and in vivo experiments suggest that Withaferin A can be used for synergistic antibacterial, antibiofilm and anti-inflammatory activity to treat infections due S. aureus.

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies.

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.