Complete genome sequence and genome-wide transposon mutagenesis enable the determination of genes required for sodium hypochlorite tolerance and drug resistance in pathogen Aeromonas veronii GD2019.

Aeromonas veronii, a significant pathogen in aquatic environments, poses a substantial threat to both human and animal health, particularly in aquaculture. In this study, we isolated A. veronii strain GD2019 from diseased largemouth bass (Micropterus salmoides) during a severe outbreak of aeromonad septicemia in Guangdong Province, China. The complete genome sequence of A. veronii GD2019 revealed that GD2019 contains a single chromosome of 4703,168 bp with an average G+C content of 58.3%. Phylogenetic analyses indicated that GD2019 forms a separate sub-branch in A. veronii and comparative genomic analyses identified the existence of an intact Type III secretion system. Moreover, to investigate the genes that are required for the conditional fitness of A. veronii under various stresses, a high-density transposon insertion library in GD2019 was generated by a Tn5-based transposon and covers 6311 genomic loci including 4155 genes and 2156 intergenic regions. Leveraging this library, 630 genes were classified as essential genes for growth in rich-nutrient LB medium. Furthermore, the genes GE001863/NtrC and GE002550 were found to confer tolerance to sodium hypochlorite in A. veronii. GE002562 and GE002614 were associated with the resistance to carbenicillin. Collectively, our results provide abundant genetic information on A. veronii, shedding light on the pathogenetic mechanisms of Aeromonas.

Unveiling novel threats: Urban river isolation of Aeromonas veronii with unusual VEB-28 extended-spectrum ß-lactamase and distinct mcr variants.

Aeromonas spp. are frequently encountered in aquatic environments, with Aeromonas veronii emerging as an opportunistic pathogen causing a range of diseases in both humans and animals. Recent reports have raised public health concerns due to the emergence of multidrug-resistant Aeromonas spp. This is particularly noteworthy as these species have demonstrated the ability to acquire and transmit antimicrobial resistance genes (ARGs). In this study, we report the genomic and phenotypic characteristics of the A. veronii TR112 strain, which harbors a novel variant of the Vietnamese Extended-spectrum ß-lactamase-encoding gene, blaVEB-28, and two mcr variants recovered from an urban river located in the Metropolitan Region of São Paulo, Brazil. A. veronii TR112 strain exhibited high minimum inhibitory concentrations (MICs) for ceftazidime (64 µg/mL), polymyxin (8 µg/mL), and ciprofloxacin (64 µg/mL). Furthermore, the TR112 strain demonstrated adherence to HeLa and Caco-2 cells within 3 h, cytotoxicity to HeLa cells after 24 h of interaction, and high mortality rates to the Galleria mellonella model. Genomic analysis showed that the TR112 strain belongs to ST257 and presented a range of ARGs conferring resistance to ß-lactams (blaVEB-28, blaCphA3, blaOXA-912) and polymyxins (mcr-3 and mcr-3.6). Additionally, we identified a diversity of virulence factor-encoding genes, including those encoding mannose-sensitive hemagglutinin (Msh) pilus, polar flagella, type IV pili, type II secretion system (T2SS), aerolysin (AerA), cytotoxic enterotoxin (Act), hemolysin (HlyA), hemolysin III (HlyIII), thermostable hemolysin (TH), and capsular polysaccharide (CPS). In conclusion, our findings suggest that A. veronii may serve as an environmental reservoir for ARGs and virulence factors, highlighting its importance as a potential pathogen in public health.

Role of Type VI secretion system in pathogenic remodeling of host gut microbiota during Aeromonas veronii infection.

Intestinal microbial disturbance is a direct cause of host disease. The bacterial Type VI secretion system (T6SS) often plays a crucial role in the fitness of pathogenic bacteria by delivering toxic effectors into target cells. However, its impact on the gut microbiota and host pathogenesis is poorly understood. To address this question, we characterized a new T6SS in the pathogenic Aeromonas veronii C4. First, we validated the secretion function of the core machinery of A. veronii C4 T6SS. Second, we found that the pathogenesis and colonization of A. veronii C4 is largely dependent on its T6SS. The effector secretion activity of A. veronii C4 T6SS not only provides an advantage in competition among bacteria in vitro, but also contributes to occupation of an ecological niche in the nutritionally deficient and anaerobic environment of the host intestine. Metagenomic analysis showed that the T6SS directly inhibits or eliminates symbiotic strains from the intestine, resulting in dysregulated gut microbiome homeostasis. In addition, we identified three unknown effectors, Tse1, Tse2, and Tse3, in the T6SS, which contribute to T6SS-mediated bacterial competition and pathogenesis by impairing targeted cell integrity. Our findings highlight that T6SS can remodel the host gut microbiota by intricate interplay between T6SS-mediated bacterial competition and altered host immune responses, which synergistically promote pathogenesis of A. veronii C4. Therefore, this newly characterized T6SS could represent a general interaction mechanism between the host and pathogen, and may offer a potential therapeutic target for controlling bacterial pathogens.

Inhibition mechanism of crude lipopeptide from Bacillus subtilis against Aeromonas veronii growth, biofilm formation, and spoilage of channel catfish flesh.

Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.

Genome-wide DNA N6-methyladenosine in Aeromonas veronii and Helicobacter pylori.

BACKGROUND: DNA N6-methyladenosine (6mA), as an important epigenetic modification, widely exists in bacterial genomes and participates in the regulation of toxicity, antibiotic resistance, and antioxidant. With the continuous development of sequencing technology, more 6mA sites have been identified in bacterial genomes, but few studies have focused on the distribution characteristics of 6mA at the whole-genome level and its association with gene expression and function. RESULTS: This study conducted an in-depth analysis of the 6mA in the genomes of two pathogenic bacteria, Aeromonas veronii and Helicobacter pylori. The results showed that the 6mA was widely distributed in both strains. In A. veronii, 6mA sites were enriched at 3' end of protein-coding genes, exhibiting a certain inhibitory effect on gene expression. Genes with low 6mA density were associated with cell motility. While in H. pylori, 6mA sites were enriched at 5' end of protein-coding genes, potentially enhancing gene expression. Genes with low 6mA density were closely related to defense mechanism. CONCLUSIONS: This study elucidated the distribution characteristics of 6mA in A. veronii and H. pylori, highlighting the effects of 6mA on gene expression and function. These findings provide valuable insights into the epigenetic regulation and functional characteristics of A. veronii and H. pylori.

Evaluate the potential use of TonB-dependent receptor protein as a subunit vaccine against Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus).

Aeromonas veronii is an emerging bacterial pathogen that causes serious systemic infections in cultured Nile tilapia (Oreochromis niloticus), leading to massive deaths. Therefore, there is an urgent need to identify effective vaccine candidates to control the spread of this emerging disease. TonB-dependent receptor (Tdr) of A. veronii, which plays a role in the virulence factor of the organism, could be useful in terms of protective antigens for vaccine development. This study aims to evaluate the potential use of Tdr protein as a novel subunit vaccine against A. veronii infection in Nile tilapia. The Tdr gene from A. veronii was cloned into the pET28b expression vector, and the recombinant protein was subsequently produced in Escherichia coli strain BL21 (DE3). Tdr was expressed as an insoluble protein and purified by affinity chromatography. Antigenicity test indicated that this protein was recognized by serum from A. veronii infected fish. When Nile tilapia were immunized with the Tdr protein, specific antibody levels increased significantly (p-value <0.05) at 7 days post-immunization (dpi), and peaked at 21 dpi compared to antibody levels at 0 dpi. Furthermore, bacterial agglutination activity was observed in the fish serum immunized with the Tdr protein, indicating that specific antibodies in the serum can detect Tdr on the bacterial cell surface. These results suggest that Tdr protein has potential as a vaccine candidate. However, challenging tests with A.veronii in Nile tilapia needs to be investigated to thoroughly evaluate its protective efficacy for future applications.

Characteristics of CD4-1 gene and its immune responses against Aeromonas veronii infection by activating NF-κB signaling in Qihe crucian carp Carassius auratus.

CD4-1 found in bony fish contains four extracellular immunoglobulin (Ig)-like domains similar to that of mammalian CD4, which is crucial for the activation of CD4+ helper T-cell. However, there is limited knowledge regarding the molecular markers, immune functions and regulation mechanism of CD4-1 in teleosts due to their vast diversity. In this study, we cloned and characterized two isoforms of Qihe crucian carp CD4-1, designated as CaCD4-1.1 and CaCD4-1.2. We further explored their expression responses upon stimulation with Aeromonas veronii, and the regulation of their immune responses against A. veronii by NF-κB. The ORF of CaCD4-1.1 and CaCD4-1.2 cDNA encoded 477 and 466 amino acids, respectively. Both proteins contained seven conserved cysteine residues in the extracellular domain, and a CCC motif in their cytoplasm, respectively. However, CaCD4-1.1 exhibited a relatively limited similarity with CaCD4-1.2 in the ectodomain. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the mRNA expression of CaCD4-1.1 and CaCD4-1.2 exhibited differential constitutive expression across all examined tissues. Furthermore, the expression level of CD4-1.2 was higher than that of CD4-1.1 in the gills, head kidney, and spleen of Qihe crucian carp subjected to A. veronii challenge, while it was lower in the trunk kidney. Inhibition of NF-κB activity resulted in a decrease in the expression levels of CD4-1.1 and CD4-1.2 mRNA in the gill, while inducing an increase in expression levels in the spleen, in accordance with the observed ultrastructural changes in both organs. Interestingly, the impact of NF-κB on the mRNA expression level of CD4-1.1 appears to be stronger than that of CD4-1.2. Our results suggest that CaCD4-1.1 and CaCD4-1.2 could be expressed on T cells and antigen-sampling cells that exhibit similar characteristics to mammalian M cells, respectively, and differentially regulated by NF-κB in adaptive immune responses against bacterial infection. This research contributes to a better understanding of the crucial role of CD4-1 in the immune response of Qihe crucian carp and provide novel insights for the prevention and treatment of fish diseases in aquaculture.

Identification and characterisation of emerging fish pathogens Aeromonas veronii and Aeromonas hydrophila isolated from naturally infected Channa punctata.

Naturally infected Channa punctata exhibiting bacterial septicemic syndrome including ulcerations along with mortality records were collected from a fish farm in Assam during winter season (early November 2020 to early January 2021). The moribund fishes were subjected for bacterial isolation followed by identification of the bacteria. Two dominant emerging bacterial pathogens were identified as Aeromonas veronii (isolate ZooGURD-01) and Aeromonas hydrophila (isolate ZooGURD-05) by standard biochemical characterization and 16S rRNA and rpo B gene amplification. Re-infection experiments of both the bacterial isolates in healthy disease-free C. punctata showed similar symptoms to that of natural infection thus confirming their virulence. The LD50 calculated during challenge test for both the isolates ZooGURD-01 and ZooGURD-05 found to be pathogenic at 2.6 × 104 and 1.6 × 104 CFU/fish respectively. Further PCR amplification of specific virulent genes (aerolysin, hemolysin and enterotoxin) confirmed pathogenicity for both isolates. Histopathological examinations of liver and kidney in re-infection experiments showed prominent changes supporting bacterial septicaemia. Antibiotic sensitivity pattern showed that the isolates ZooGURD-01 and ZooGURD-05 were sensitive to 22 and 19 out of 25 antimicrobials respectively. The present study was the first report on the mortality of farmed C. punctata associated with natural infection caused by A. veronii and A. hydrophila with no record of pathogenicity of A. veronii in C. punctata.

Characteristics and Complete Genome Analysis of a Pathogenic Aeromonas Veronii SJ4 from Diseased Siniperca Chuatsi.

As an opportunistic pathogen, Aeromonas veronii can cause hemorrhagic septicemia of various aquatic animals. In our present study, a dominant strain SJ4, isolated from naturally infected mandarin fish (Siniperca chuatsi), was identified as A. veronii according to the morphological, physiological, and biochemical features, as well as molecular identification. Intraperitoneal injection of A. veronii SJ4 into S. chuatsi revealed clinical signs similar to the natural infection, and the median lethal dosage (LD50) of the SJ4 to S. chuatsi in a week was 3.8 × 105 CFU/mL. Histopathological analysis revealed that the isolate SJ4 could cause cell enlargement, obvious hemorrhage, and inflammatory responses in S. chuatsi. Detection of virulence genes showed the isolate SJ4 carried act, fim, flgM, ompA, lip, hly, aer, and eprCAL, and the isolate SJ4 also produce caseinase, dnase, gelatinase, and hemolysin. In addition, the complete genome of A. veronii SJ4 was sequenced, and the size of the genome of A. veronii SJ4 was 4,562,694 bp, within a G + C content of 58.95%, containing 4079 coding genes. Nine hundred ten genes encoding for several virulence factors, such as type III and VI secretion systems, flagella, motility, etc., were determined based on the VFDB database. Besides, 148 antibiotic resistance-related genes in 27 categories related to tetracyclines, fluoroquinolones, aminoglycosides, macrolides, chloramphenicol, and cephalosporins were also annotated. The present results suggested that A. veronii was etiological agent causing the bacterial septicemia of S. chuatsi in this time, as well as provided a valuable base for revealing pathogenesis and resistance mechanism of A. veronii.

Pathogenicity and inactivated vaccine treatment of Aeromonas veronii JW-4 on crucian carp.

Aeromonas veronii is a common bacterium found in a variety of aquatic environments, capable of causing a diverse array of diseases in both aquatic animals and humans. Therefore, evaluating the pathogenicity of A. veronii and implementing measures to control its spread are essential. In this study, a strain JW-4, identified as A. veronii, was isolated from diseased Scaphesthes macrolepis, a grade Ⅱ protected animal in China. To investigate the pathogenicity of the strain, fish were fed with serial levels JW-4 supplemented diet or basal diet (control group 1, CG1) for 28 days (d). Results showed that JW-4 stimulated an immune response, evidenced by an increase in immune-related enzyme activities (GOT and GPT) of serum and liver and an upregulation of genes expression levels (TNF-α and IFN-γ) of liver and spleen, and these effects gradually decreased over time. Histopathological examination revealed that JW-4 could alter the tissue structure of immune organs, such as liver and kidney. These changes were accompanied by vacuolar degeneration, nuclear dissolution, and an increased lymphocyte count. To assess protective effects of a vaccine against this strain, fish were injected with an inactivated vaccine (immunization group, IG) or 0.85% sterile saline (control group 2, CG2) for 28-day observation period, then challenged with JW-4 on the 28th day. The inactivated vaccine enhanced total and specific IgM to A. veronii levels of the fish, resulting in a relative percentage survival of 75% in IG. These findings provide a foundation for identifying pathogenic bacteria and developing more effective prophylactic strategies in aquaculture.

The Dynamics of Gene Expression Unraveling the Immune Response of Macrobrachium rosenbergii Infected by Aeromonas veronii.

To further investigate the immune response of Macrobrachium rosenbergii against Aeromonas veronii, comparative transcriptomic analyses of the M. rosenbergii hepatopancreas were conducted on challenge and control groups at 6, 12, and 24 h post-infection (hpi), independently. A total of 51,707 high-quality unigenes were collected from the RNA-seq data, and 8060 differentially expressed genes (DEGs) were discovered through paired comparisons. Among the three comparison groups, a KEGG pathway enrichment analysis showed that 173 immune-related DEGs were considerably clustered into 28 immune-related pathways, including the lysosome, the phagosome, etc. Moreover, the expression levels of the four key immune-related genes (TOLL, PAK1, GSK3ß, and IKKα) were evaluated at various stages following post-infection in the hepatopancreas, hemolymph, and gills. Both PAK1 and GSK3ß genes were highly up-regulated in all three tissues at 6 hpi with A. veronii; TOLL was up-regulated in the hepatopancreas and hemolymph but down-regulated in the gill at 6 hpi, and IKKα was up-regulated in hemolymph and gill, but down-regulated in the hepatopancreas at 6 hpi. These findings lay the groundwork for understanding the immune mechanism of M. rosenbergii after contracting A. veronii.

Development of silver nano-based indirect ELISA and Dot-ELISA methods for serological diagnosis of a bacterial fish pathogen Aeromonas veronii.

Rapid and accurate detection of bacterial pathogens is critical in controlling disease outbreaks affecting farmed fish. The present study aimed to develop a novel serological diagnostic approach using nano­silver based Enzyme-linked immunosorbent assay (ELISA) for speedy detection of Aeromonas veronii infections in Nile tilapia. A. veronii isolates used in ELISA assays were recovered from moribund Nile tilapia during a disease outbreak in a private fish farm in Egypt. A. veronii isolates were identified based on alignment analysis of the gyrB and 16S rRNA gene sequences. A. veronii antisera used in ELISA assays were prepared in tilapia, and the bacterial antigens were formalin-killed. The cut-off values were 0.46 and 0.48 in traditional and nano-based ELISA. There were no cross-reactions with bacterial isolates (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas fluorescens, and Vibrio vulnificus). The lowest antigen concentration that produced positive results after checkerboard titration in indirect-ELISA (i-ELISA) and dot ELISA was 15 µg and 250 ng of prepared antigen, respectively. Nano-ELISA and nano-based dot-ELISA antigen concentration was 10 µg and 100 ng, respectively. Sera concentration was 1:100 in indirect-ELISA and dot-ELISA, while it was 1:50 in nano-based ELISA and nano dot-ELISA. The i-ELISA successfully detected anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity, while in the dot-ELISA, the sensitivity and specificity were 83.33% and 100%, respectively. Nano dot-ELISA had 100% sensitivity, specificity, and accuracy. Nano dot-ELISA assays have higher specificity, sensitivity, and accuracy than traditional ELISAs in detecting A. veronii. Further studies are needed to develop a rapid test kit for on-site field diagnosis.

OmpR (TCS response regulator) of Aeromonas veronii plays a major role in drug resistance, stress resistance and virulence by regulating biofilm formation.

Aeromonas veronii (A. veronii), a highly pathogenic bacteria with a wide range of hosts, widely exists in the environment of humans, animals and aquatic animals, and can cause a variety of diseases. In this study, the receptor regulator ompR in the envZ/ompR of two-component system was selected to construct a mutant strain (Δ ompR) and a complement strain (C-ompR) to explore the regulatory effect of ompR on the biological characteristics and virulence of TH0426. The results showed that the ability of biofilm formation and osmotic stress of TH0426 were significantly reduced (P < 0.001), the resistance to ceftriaxone and neomycin were slightly down-regulate when the ompR gene was deleted. At the same time, animal pathogenicity experiments showed that the virulence of TH0426 was significantly down-regulated (P < 0.001). These results indicated that ompR gene regulates the biofilm formation of TH0426, and regulates some biological characteristics of TH0426, including drug sensitivity, resistance to osmotic stress, and also affects its virulence.

Genomic traits of Aeromonas veronii isolated from slaughtered Danish broilers.

Aeromonas veronii is a Gram-negative opportunistic bacterium found in fish, poultry and humans and has occasionally been associated with disease although not generally considered a poultry pathogen. A. veronii was recently isolated from both healthy and condemned broiler carcasses at a major Danish abattoir. In this study, we did a whole genomic analysis of 24A. veronii strains from the abattoir to determine their potential sources and relatedness as well as their pathogenic potential, antimicrobial resistance determinants and associated mobile elements. No strains were multi-drug resistant, but all strains carried the beta-lactam resistance genes cphA3 and blaOXA-12 without being phenotypically resistant to carbapenems. One strain carried an IncA plasmid with tet(A), tet(B) and tet(E) genes. A phylogenetic tree including public A. veronii sequences showed that our isolates were not clonal but were dispersed around the phylogenetic tree, suggesting a diffuse spread of A. veronii across human, aquatic and poultry samples. Strains carried different virulence factors known to be associated with pathogenesis and severity of disease in animals and humans, e.g. type II (aerolysin, amylases, proteases, and cytotoxic enterotoxin Act) and III secretion systems where the latter has been associated with mortality in hospitalized patients. Although our genomic analysis of A. veronii shows zoonotic potential, epidemiological studies of human gastro-enteritis cases of A. veronii associated with consumption of broiler meat are needed. It remains to be proven if A. veronii is a true poultry pathogen and part of the established microflora in abattoirs and the gut-intestinal microflora of poultry.

Concurrent infections of Streptococcus iniae and Aeromonas veronii in farmed Giant snakehead (Channa micropeltes).

The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram-positive cocci and cream-coloured, round and convex colonies of rod-shaped gram-negative bacteria. Additional biochemical and species-specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole-trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole-trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies.

Transcriptomic insights into the immune response of the intestine to Aeromonas veronii infection in northern snakehead (Channa argus).

Intestinal inflammation is a protective response that is implicated in bacterial enteritis triggered by gastrointestinal infection. The immune mechanisms elicited in teleost against the infection of Aeromonas veronii are largely unknown. In this study, we performed a de novo northern snakehead (Channa argus) transcriptome assembly using Illumina sequencing platform. On this basis we performed a comparative transcriptomic analysis of northern snakehead intestine from A. veronii-challenge and phosphate buffer solution (PBS)-challenge fish, and 2076 genes were up-regulated and 1598 genes were down-regulated in the intestines infected with A. veronii. The Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes (DEGs) were enriched to 27, 21 and 20 GO terms in biological process, cellular component, and molecular function, respectively. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 420 DEGs were involved in 194 pathways. Moreover, 33 DEGs were selected for quantitative real-time PCR analysis to validate the RNA-seq data. The results reflected the consistency of the expression levels between qRT-PCR and RNA-seq data. In addition, a time-course analysis of the mRNA expression of 33 immune-related genes further indicated that the intestinal inflammation to A. veronii infection simultaneously regulated gene expression alterations. The present study provides transcriptome data of the teleost intestine, allowing us to understand the mechanisms of intestinal inflammation triggered by bacterial pathogens. DATA AVAILABILITY STATEMENT: All data supporting the findings of this study are available within the article and Supplementary files. The RNA-seq raw sequence data are available in NCBI short read archive (SRA) database under accession number PRJNA615958.

Construction and efficacy of Aeromonas veronii mutant Δhcp as a live attenuated vaccine for the largemouth bass (Micropterus salmoides).

Aeromonas veronii is a human and animal co-pathogenic bacterium that could have a significant negative impact on both human health and aquaculture. In this study, a mutant strain of A. veronii with deletion of the hemolysin co-regulated protein (hcp) gene was constructed (Δhcp-AV). Compared with the wild strain, Δhcp-AV showed significantly reduced growth capacity and biofilm formation ability. Motility tests showed that the hcp gene had no significant effect on the swimming and swarming ability. In addition, the pathogenicity was also reduced. To evaluate the efficacy of Δhcp-AV as a live attenuated vaccine for prevention of Aeromonas veronii infection, we compared the immune response of largemouth bass (Micropterus salmoides) after immunization with 500 µL of 1.47 × 105 CFU/mL of Δhcp-AV and 4 × 108 CFU/mL of inactivated A. veronii. Obvious increases of serum immune related enzyme activity were observed in immunization groups. Expression levels of immune-related genes in Δhcp-AV group were up-regulated, and higher than those in inactivated A. veronii group. After challenging with live A. veronii, the relative percent survival (RPS) was 100% in Δhcp- AV group, whereas the RPS was 76.67% in inactivated A. veronii group. Our data suggest that the live attenuated vaccine Δhcp- AV could elicit a stronger immune response and provide a higher RPS than inactivated A. veronii. These data suggest that hcp gene is an important virulence factor of A. veronii, and the live attenuated vaccine Δhcp-AV is safe and effective for prevention A. veronii infection in M. salmoides farming.

Dual RNA-seq of spleens extracted from channel catfish infected with Aeromonas veronii reveals novel insights into host-pathogen interactions.

Interactions between host and pathogen are involving various dynamic changes in transcript expression and critical for understanding host immunity against infections and its associated pathogenesis. Herein, we established a model of channel catfish infected with Aeromonas veronii. The infected fish had prominent body surface bleeding, and the spleen showed hyperemia and swelling. Then, the spleen of channel catfish infected with A. veronii was analyzed by dual RNA sequencing (RNA-seq), and the transcriptome data were compared with uninfected channel catfish spleen or bacteria cultured in vitro. The transcript expression profile of pathogen-host interaction between A. veronii and channel catfish was successfully studied. During infection, the host was enriched for multiple immune-related signaling pathways, such as the Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, and T cell receptor signaling pathway; and significantly upregulated for many innate immune-related genes, including IL-8. At the same time, we found that A. veronii mainly harmed the host spleen through hemolysin. Our current findings are of great significance in clarifying the pathogenesis of channel catfish induced by A. veronii and provide gene targets for developing preventive measures.

Characterization of CD3γ/δ gene and its immune response in Qihe crucian carp Carassius auratus after challenged by Aeromonas veronii and Poly(I:C).

CD3γ/δ found in non-mammalian vertebrates is a CD3 homolog with structural characteristics similar to both mammalian CD3γ and CD3δ, and plays important roles in T cell recognization and immune response in fish. In this study, the full-length of CD3γ/δ from Qihe crucian carp (named CaCD3γ/δ) was cloned and characterized, then the expression response profiles and potential immune functions was explored after Aeromonas veronii and Poly(I:C) challenge. The results showed that the full-length of CaCD3γ/δ was 819 bp including a 5'-UTR of 141 bp, a 3'-UTR of 168 bp, and an ORF of 510 bp encoding a putative 169-aa protein with an estimated MW of 18.71 kD and a theoretical pI of 8.77. The protein sequence of CaCD3γ/δ contained a Leu-Leu and a CXXXC motif in the extracellular domain, and an ITAM and a Leu-Ile motif in the cytoplasm, and a residue of Asn in the transmembrane. CaCD3γ/δ was constitutively expressed in the spleen, liver, gill, and blood of Qihe crucian carp. After the carp were challenged with Poly(I:C) and Aeromonas veronii, the mRNA expression levels of CaCD3γ/δ were significantly changed in the spleen, head kidney, intestine and gill, according to the results of qPCR. However, compared with A. veronii, Poly(I:C) challenge can rapidly induce the CaCD3γ/δ expression levels in head kidney, intestine and spleen, which suggested CaCD3γ/δ may be differentially modulated by different pathogens. Moreover, the results of immunohistochemical analysis showed that the CaCD3γ/δ+ secreted cells in the spleen and gills of Qihe crucian were increased after challenged with Poly(I:C), as well as the spleen challenged with A. veronii, but at different levels. Combined with the fact that vascular congestion, necrosis of parenchymal cells, and inflammatory cells including lymphocytes infiltration were also observed in the gill and spleen of Qihe crucian carp treated with A. veronii and Poly(I:C) revealed by pathological analysis, it was predicted that CaCD3γ/δ+ T lymphocytes may participated in the immune response against pathogens. This study will contribute to understand the important role of CaCD3γ/δ+ T lymphocytes in the immune response of Qihe crucian carp, and provide new insights for the prevention and treatment of the diseases of Qihe crucian carp.

Severe skin and soft tissue infection in the left upper limb caused by Aeromonas veronii: a case report.

INTRODUCTION: Skin and soft tissue infections are common because of exposure to aquatic environment, while severe infections caused by Aeromonas veronii are rare. CASE PRESENTATION: We report a case of severe skin and soft tissue infection of the left upper limb caused by Aeromonas veronii. A 50-year-old Chinese woman, who had a history of cardiac disease and type 2 diabetes mellitus, accidentally injured her left thumb while cutting a fish. Early antibiotic therapy and surgical debridement was performed before the result of bacterial culture came back. Whole-genome sequencing was further performed to confirm the pathogen and reveal the drug resistance and virulence genes. The wound was gradually repaired after 1 month of treatment, and the left hand recovered well in appearance and function after 3 months of rehabilitation. CONCLUSION: Early diagnosis, surgical intervention, and administration of appropriate antibiotics are crucial for patients who are suspected of having skin and soft tissue infection, or septicemia caused by Aeromonas veronii.