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Fish & Shellfish Immunology Sep 2019Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex...
Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (△Hem, △TS1, △TS2, △TS1△TS2, and △II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.
Topics: Animals; Bacterial Vaccines; Catfishes; Fish Diseases; Gene Editing; Gene Knockout Techniques; Vaccines, Attenuated; Vibrio Infections; Vibrio mimicus
PubMed: 31202969
DOI: 10.1016/j.fsi.2019.06.025 -
Microbial Pathogenesis Aug 2023Type II secretion systems (T2SS) are important molecular machines used by bacteria to transport a wide range of proteins across the outer membrane from the periplasm....
Type II secretion systems (T2SS) are important molecular machines used by bacteria to transport a wide range of proteins across the outer membrane from the periplasm. Vibrio mimicus is an epidemic pathogen threats to both aquatic animals and human health. Our previous study demonstrates that T2SS deletion reduced virulence by 307.26 times in yellow catfish. However, the specific effects of T2SS-mediated extracellular protein secretion in V. mimicus, including its potential role in exotoxin secretion or other mechanisms, require further investigation. Through proteomics and phenotypic analyses, this study observed that the ΔT2SS strain exhibited significant self-aggregation and dynamic deficiency, with a notable negative correlation with subsequent biofilm formation. The proteomics analysis revealed 239 different abundances of extracellular proteins after T2SS deletion, including 19 proteins with higher abundance and 220 proteins with lower and even absent in the ΔT2SS strain. These extracellular proteins are involved in various pathways, such as metabolism, virulence factors expression, and enzymes. Among them, purine, pyruvate, and pyrimidine metabolism, and the Citrate cycle, were the primary pathways affected by T2SS. Our phenotypic analysis is consistent with these findings, suggesting that the decreased virulence of ΔT2SS strains is due to the effect of T2SS on these proteins, which negatively impacts growth, biofilm formation, auto-aggregation, and motility of V. mimicus. These results provide valuable insights for designing deletion targets for attenuated vaccines development against V. mimicus and expand our understanding of the biological functions of T2SS.
Topics: Animals; Humans; Type II Secretion Systems; Vaccines, Attenuated; Bacterial Proteins; Virulence; Virulence Factors
PubMed: 37380063
DOI: 10.1016/j.micpath.2023.106215 -
Fish & Shellfish Immunology Apr 2024Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense, leading to high mortality and financial loss. Based on previous...
Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense, leading to high mortality and financial loss. Based on previous studies, rpoS gene contribute to bacterial pathogenicity during infection, but the role of RpoS involved in the immune response of M. nipponense under V. mimicus infection remains unclear. In this study, the pathogen load and the RNA-seq of M. nipponense under wild-type and ΔrpoS strain V. mimicus infection were investigated. Over the entire infection period, the ΔrpoS strain pathogen load was always lower than that of the wild-type strain in the M. nipponense hemolymph, hepatopancreas, gill and muscle. Furthermore, the expression level of rpoS gene in the hepatopancreas was the highest at 24 hours post infection (hpi), then the samples of hepatopancreas tissue infected with the wild type and ΔrpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with ΔrpoS strain. In contrast to the wild-type infected group, the ΔrpoS strain infected group exhibited differentially expressed genes (DEGs) enriched in 181 KEGG pathways at 24 hpi. Among these pathways, 8 immune system-related pathways were enriched, including ECM-receptor interaction, PI3K-Akt signaling pathway, Rap1 signaling pathway, Gap junction, and Focal adhesion, etc. Among these pathways, up-regulated genes related to Kazal-type serine protease inhibitors, S-antigen protein, copper zinc superoxide dismutase, tight junction protein, etc. were enriched. This study elucidates that rpoS can affect tissue bacterial load and immune-related pathways, thereby impacting the survival rate of M. nipponense under V. mimicus infection. These findings validate the potential of rpoS as a promising target for the development of a live attenuated vaccine against V. mimicus.
Topics: Animals; Palaemonidae; Vibrio mimicus; Phosphatidylinositol 3-Kinases; Transcriptome; Vibrio Infections; Immunity
PubMed: 38342414
DOI: 10.1016/j.fsi.2024.109440 -
MSphere Aug 2021Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a...
Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.
Topics: Bacterial Proteins; Caco-2 Cells; Computational Biology; Gene Expression Regulation, Bacterial; Humans; Interleukin-8; Multigene Family; Protein Serine-Threonine Kinases; Protein Transport; Serine; Type III Secretion Systems; Vibrio parahaemolyticus
PubMed: 34346702
DOI: 10.1128/mSphere.00599-21 -
Journal of Applied Microbiology Jul 2020The current study was conducted to determine the incidence, antibiotic resistance and virulence genes of Vibrio strains isolated from ready-to-eat shrimps in Edo and...
AIM
The current study was conducted to determine the incidence, antibiotic resistance and virulence genes of Vibrio strains isolated from ready-to-eat shrimps in Edo and Delta States, Nigeria.
METHODS AND RESULTS
A total of 1440 ready-to-eat shrimp samples were obtained from open markets from November 2016 to October 2017 and analysed using standard culture-based procedures. Overall, our result showed that the ready-to-eat shrimp samples had high mean aerobic mesophilic bacterial count between 3·543 and 7·489 log CFU per gram. Vibrio cell densities ranged between 0·663 and 6·761 log CFU per gram. From the total samples, 1343/1440 (93·3%) were positive for Vibrio species where 120 Vibrio isolates were randomly selected and confirmed using genus- and species-specific PCR approach. The PCR identification revealed the presence of Vibrio parahaemolyticus 46 (38·33%), Vibrio vulnificus 14 (11·67%), Vibrio fluvialis 12 (10%), Vibrio alginolyticus 8 (6·67%), Vibrio cholerae 2 (1·67%), Vibrio mimicus 10 (8·33%), Vibrio harveyi 3 (2·5%) and other Vibrio sp. 25 (20·83%). All Vibrio isolates were sensitive to colistin and gentamycin with varying percentage of resistance to other antibiotics used in the study. Multiple antibiotic-resistant (MAR) index ranged from 0·08 to 0·83. The tcp, tdh and trl virulence genes were identified in 95 (79·2%), 92 (76·7%) and 95 (79·2%) of the examined isolates respectively. Antibiotic-resistant genes also revealed the presence of class 1 integrase 75 (62·5%), sul2 87 (72·5%), strB 94 (78·3%) and catB3 68 (56·7%).
CONCLUSION
Our findings revealed that the ready-to-eat shrimps may serve as potential reservoirs and medium in the dissemination of prospective MAR pathogens to the consumers and thus constitute a potential risk to public health.
SIGNIFICANCE AND IMPACT OF THE STUDY
The findings from this study represent the first comprehensive report of Vibrio isolates from ready-to-eat shrimps in Edo and Delta States, Nigeria. Incessant monitoring of Vibrio strains and their predisposition to antimicrobials is a necessity to guarantee seafood safety and guarantee the best treatment regimen for patients with gastroenteritis.
Topics: Animals; Anti-Bacterial Agents; Drug Resistance, Microbial; Genes, Bacterial; Microbial Sensitivity Tests; Nigeria; Shellfish; Vibrio; Virulence Factors
PubMed: 31977129
DOI: 10.1111/jam.14590 -
Applied and Environmental Microbiology Apr 2021By characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that...
By characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that influence this spread of resistance. Our aim was to investigate the host network of a multidrug resistance broad-host-range plasmid in the culturable gut microbiome of zebrafish. This was done through and conjugation experiments with as the donor of the plasmid pB10:: When this donor was mixed with the extracted gut microbiome, only transconjugants of were detected. In separate matings between the same donor and four prominent isolates from the gut microbiome, the plasmid transferred to two of these four isolates, and , but not to and When these and transconjugants were the donors in matings with the same four isolates, the plasmid now also transferred from to was unable to donate the plasmid, and was unable to acquire it. Finally, when the donor was added to zebrafish through their food, plasmid transfer was observed in the gut, but only to , a rare member of the gut microbiome. This work shows that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome depends on the donor-recipient species combinations and therefore their spatial arrangement. It also suggests that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food. To understand how antibiotic resistance plasmids end up in human pathogens, it is crucial to learn how, where, and when they are transferred and maintained in members of bacterial communities such as the gut microbiome. To gain insight into the network of plasmid-mediated antibiotic resistance sharing in the gut microbiome, we investigated the transferability and maintenance of a multidrug resistance plasmid among the culturable bacteria of the zebrafish gut. We show that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome can depend on which species are involved, as some are important nodes in the plasmid-host network and others are dead ends. Our findings also suggest that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.
Topics: Animals; Bacteria; Drug Resistance, Multiple, Bacterial; Female; Gastrointestinal Microbiome; Male; Plasmids; Zebrafish
PubMed: 33637574
DOI: 10.1128/AEM.02735-20 -
Frontiers in Physiology 2019A feeding trial was carried out to evaluate the effects of substitution of fishmeal (FM) by dietary poultry by-product meal, fermented by and on growth, intestinal...
A feeding trial was carried out to evaluate the effects of substitution of fishmeal (FM) by dietary poultry by-product meal, fermented by and on growth, intestinal health, microbial composition, immune related cytokines and disease resistance of freshwater crayfish, marron () against Two isonitrogenous and isocaloric diets were formulated by replacing FM protein with fermented poultry by-product meal (FPBM) protein at 0% (Control) and 75% (FPBM), and fed marron for 70 days. The results indicated no significant difference ( > 0.05) in final body weights between two groups of marron, whilst intestinal microvilli number per fold was increased in marron fed FPBM than the control. The 16S rRNA sequences revealed an increased number of and , and decreased number of at genus level in the distal intestine of marron fed FPBM. Marron fed FPBM showed up-regulated expression of IL-8, IL-10, and IL-17F genes in the distal intestine. Significantly ( < 0.05) increased lysozyme and phagocytic activity, and higher survival was found in marron fed FPBM following a bacterial challenge with . Therefore, it is concluded that FPBM is beneficial to marron in terms of microbial community, immune-related cytokines and disease resistance against
PubMed: 32082185
DOI: 10.3389/fphys.2019.01635 -
Molecular and Cellular Probes Feb 2021A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and...
A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5-50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.
Topics: Electrophoresis, Capillary; Estuaries; Humans; Multiplex Polymerase Chain Reaction; Plesiomonas; Sensitivity and Specificity; Vibrio; Water Microbiology
PubMed: 33338586
DOI: 10.1016/j.mcp.2020.101689 -
Virulence Dec 2020is a foodborne pathogen, which is widely distributed in the aquatic environment. Moreover, it is often involved in aquatic animal diseases. In recent years, is an...
is a foodborne pathogen, which is widely distributed in the aquatic environment. Moreover, it is often involved in aquatic animal diseases. In recent years, is an emerging pathogen in some species of Siluriformes. The strain SCCF01 was isolated from yellow catfish (). In this study, we aimed to perform genomic analysis of strain SCCF01 to identify genetic features and evolutionary relationships. Information on gene function and classification was obtained by functional annotation, and circular graph of strain SCCF01 genome, which was created by Circos v0.64. Information on virulence genes (adhesion, flagellum system, exotoxin, and secretory system, etc.) was obtained by virulence genes annotation. Genome element prediction showed that most of the mobile elements were distributed in chromosome I. Therefore, chromosome I of SCCF01 genome has more plasticity than chromosome II and might be larger in size. Genomic linear relationship between the strain of s and strain SCCF01 was analyzed by linear pairwise comparison but was unable to determine the relationship. Gene family analysis predicted that the evolutionary direction of strain SCCF01 was: clinical strain → environmental strain → SCCF01 strain. Phylogenetic analysis showed that the strain SCCF01 was more closely related to environmental strains. According to gene family analysis and phylogenetic analysis, we speculated that strain SCCF01 has probably diverged from environmental strains.
Topics: Animals; Bacterial Adhesion; Catfishes; Exotoxins; Flagella; Fresh Water; Genes, Bacterial; Genomics; Interspersed Repetitive Sequences; Phylogeny; Vibrio mimicus; Virulence; Virulence Factors
PubMed: 31826705
DOI: 10.1080/21505594.2019.1702797 -
Fish & Shellfish Immunology Mar 2022Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with...
Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with broad-spectrum antibacterial activity in fish. In mammals, some evidences demonstrated that interleukin-1β (IL-1β) primarily promote AMPs expression via activating classical NF-κB pathway, but it still remains unclear in fish. Here, the temporal and spatial expression patterns of grass carp IL-1β (gcIL-1β) gene and two AMPs genes (gchepcidin and gcβ-defensin1) in tissues post-V. mimicus infection and anti-V. mimicus activity of these two AMPs in vitro were detected, showing that V. mimicus infection significantly elevated the mRNA levels of these three genes in the immune-related tissues although their expression patterns were not entirely consistent, and both gcHepcidin and gcβ-Defensin1 possessed anti-V. mimicus activity in vitro. Subsequently, the recombinant gcIL-1β (rgcIL-1β) was expressed prokaryotically in an inclusion body, which could promote proliferation of grass carp head kidney leukocytes (gcHKLs) and enhance respiratory burst activity and phagocytic activity of head kidney macrophages. Stimulation with rgcIL-1β was able to significantly regulate the mRNA expression of key regulatory genes (il-1RI, traf6, tak1, ikkβ, iκBα and p65) involved in the activation of classical NF-κB pathway, and then induce gcTAK1 phosphorylation, promote gcp65 nuclear translocation and enhance endogenous gcIL-1β expression at both mRNA and protein levels, implying NF-κB pathway was activated. More importantly, exogenous rgcIL-1β stimulation also significantly up-regulated both gcHepcidin and gcβ-Defensin1 mRNA levels against V. mimicus, and the regulatory effect was blocked or inhibited by NF-κB inhibitor PDTC. Taken together, our results demonstrated for the first time that grass carp IL-1β stimulation could significantly enhance the expression of these two anti-V.mimicus AMPs via activating classical NF-κB pathway.
Topics: Animals; Antimicrobial Peptides; Carps; Fish Diseases; Fish Proteins; Interleukin-1beta; Mammals; NF-kappa B; Signal Transduction; Vibrio mimicus
PubMed: 34922017
DOI: 10.1016/j.fsi.2021.12.020