Analysis of global Aeromonas veronii genomes provides novel information on source of infection and virulence in human gastrointestinal diseases.

BACKGROUND: Aeromonas veronii is a Gram-negative rod-shaped motile bacterium that inhabits mainly freshwater environments. A. veronii is a pathogen of aquatic animals, causing diseases in fish. A. veronii is also an emerging human enteric pathogen, causing mainly gastroenteritis with various severities and also often being detected in patients with inflammatory bowel disease. Currently, limited information is available on the genomic information of A. veronii strains that cause human gastrointestinal diseases. Here we sequenced, assembled and analysed 25 genomes (one complete genome and 24 draft genomes) of A. veronii strains isolated from patients with gastrointestinal diseases using combine sequencing technologies from Illumina and Oxford Nanopore. We also conducted comparative analysis of genomes of 168 global A. veronii strains isolated from different sources. RESULTS: We found that most of the A. veronii strains isolated from patients with gastrointestinal diseases were closely related to each other, and the remaining were closely related to strains from other sources. Nearly 300 putative virulence factors were identified. Aerolysin, microbial collagenase and multiple hemolysins were present in all strains isolated from patients with gastrointestinal diseases. Type III Secretory System (T3SS) in A. veronii was in AVI-1 genomic island identified in this study, most likely acquired via horizontal transfer from other Aeromonas species. T3SS was significantly less present in A. veronii strains isolated from patients with gastrointestinal diseases as compared to strains isolated from fish and domestic animals. CONCLUSIONS: This study provides novel information on source of infection and virulence of A. veronii in human gastrointestinal diseases.

Long-term effects of three compound probiotics on water quality, growth performances, microbiota distributions and resistance to Aeromonas veronii in crucian carp Carassius auratus gibelio.

Probiotics could promote the healthy growth of aquatic animals and have been widely used in aquaculture. However, the influence of high concentration compound probiotics on the aquatic animals has not been reported. In the present study, a compound probiotics was used in high-density culture of crucian carps under the condition of micro-water exchange. During nearly 7-weeks feeding experiment, the aquaculture water quality, growth performances, disease resistance and microbiota distributions of crucian carps were tested. Under the high concentrations of compound probiotics, the content of total ammonia nitrogen and nitrite were finally in a state of dynamic equilibrium. The body length and weight of crucian carps in the experimental group (E) was significantly higher than that in the recirculating group (R). The antioxidant enzymes in the intestines and gills of the E group including SOD, CAT, GSH and MDA, were significantly higher than those in R group. The mortality of crucian carps in E group was significantly lower after the immersion infection of Aeromonas veronii. The addition of compound probiotics significantly increased the number of microorganisms detected in the intestines and gills of crucian carps in E group. The bacteria including Firmicutes, Planctomycetes, Verrucomicrobiota at the phylum level in E group were higher than those in R group. At the genus level, these bacteria (Pirellula, Roseimicrobium, Malikia) were not only higher in E group water, but also significantly higher in the intestines and gills than R group. The results of present study systematically analyzed the impact of high-concentration probiotics on crucian carps breeding, and speculated genus Pirellula, Roseimicrobium, Malikia may be used as aquatic probiotics. The present study will provide a new idea for the green and sustainable development of aquaculture.

Isolation of a new Streptomyces virginiae W18 against fish pathogens and its effect on disease resistance mechanism of Carassius auratus.

The Streptomyces virginiae strain W18 was screened from soil, which exhibited broad-spectrum antibacterial activity against fish pathogens. Safety assays showed that strain W18 had no toxicity to fish. Additionally, strain W18 promoted the growth performance of Carassius auratus after feeding in feed mixed with bacteria for one month. Moreover, the activities of AKP, ACP, and SOD in the serum of C. auratus were significantly increased, while the activity of LZM did not greatly change. To detect the expression levels of the genes related to immune factors in the livers, kidneys, and spleens of C. auratus, qRT-PCR was performed. The expression levels of KEAP1, IL-8, TNF-α, IL-ß, and C3 were upregulated in all three organs compared to the control, but LZM expression was downregulated in the kidney. The challenge experiment illustrated that the probability of infection with Aeromonas veronii was reduced by 60% and 40% when C. auratus was fed with two different doses of strain W18 in advance. The whole genome of strain W18 was sequenced, and the gene clusters of secondary metabolites in strain W18 were analyzed by AntiSMASH. The results showed that strain W18 contained a total of 26 gene clusters, and functional annotation analysis was conducted by using the non-coding databases COG and KEGG. All of the above results indicated that the use of strain W18 as a feed additive could enhance the resistance of C. auratus toward pathogenic bacteria and disease. In conclusion, an antagonistic strain (W18) against fish pathogenic bacteria was obtained in this study, which is of great significance for finding new treatment methods for bacterial diseases in the aquaculture industry.

Network-based analysis of virulence factors for uncovering Aeromonas veronii pathogenesis.

BACKGROUND: Aeromonas veronii is a bacterial pathogen in aquaculture, which produces virulence factors to enable it colonize and evade host immune defense. Given that experimental verification of virulence factors is time-consuming and laborious, few virulence factors have been characterized. Moreover, most studies have only focused on single virulence factors, resulting in biased interpretation of the pathogenesis of A. veronii. RESULTS: In this study, a PPI network at genome-wide scale for A. veronii was first constructed followed by prediction and mapping of virulence factors on the network. When topological characteristics were analyzed, the virulence factors had higher degree and betweenness centrality than other proteins in the network. In particular, the virulence factors tended to interact with each other and were enriched in two network modules. One of the modules mainly consisted of histidine kinases, response regulators, diguanylate cyclases and phosphodiesterases, which play important roles in two-component regulatory systems and the synthesis and degradation of cyclic-diGMP. Construction of the interspecies PPI network between A. veronii and its host Oreochromis niloticus revealed that the virulence factors interacted with homologous proteins in the host. Finally, the structures and interacting sites of the virulence factors during interaction with host proteins were predicted. CONCLUSIONS: The findings here indicate that the virulence factors probably regulate the virulence of A. veronii by involving in signal transduction pathway and manipulate host biological processes by mimicking and binding competitively to host proteins. Our results give more insight into the pathogenesis of A. veronii and provides important information for designing targeted antibacterial drugs.

Virulence and antimicrobial resistance potential of Aeromonas spp. associated with shellfish.

Aeromonas spp. are associated with seafood-related outbreaks worldwide. In seafood industry, shellfish play a major role in global seafood production. With this emerging trend of shellfish consumption, shellfish-related bacterial infections are being reported frequently. Aeromonas spp. are natural contaminants found in shellfish. Although 36 species have been identified, some species including Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria have dragged major attention as foodborne pathogenic bacteria. The ability to elaborate a variety of virulence factors of Aeromonas spp. contributes to the pathogenic activities. Also, emerging antimicrobial resistance in Aeromonas spp. has become a huge challenge in seafood industry. Furthermore, multidrug resistance increases the risk of consumer health. Studies have supplied pieces of evidence about the emerging health risk of Aeromonas spp. isolated from seafood. Therefore, the present review was intended to highlight the prevalence, virulence and antimicrobial resistance of Aeromonas spp. isolated from various types of shellfish.

Dietary Administration of Probiotic Aeromonas veronii V03 on the Modulation of Innate Immunity, Expression of Immune-Related Genes and Disease Resistance Against Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio).

This study investigates the effects of dietary Aeromonas veronii V03 supplementation on growth performances, innate immunity, and expression of immune-related genes in lymphoid organs of Cyprinus carpio and resistance to Aeromonas hydrophila infection. Fish were fed for 4 weeks with basal diet (BD; without probiotic), and experiment diet containing different doses of A. veronii V03 at 3.2 × 107 (DI) and 3.5 × 109 (DII) CFU g-1 of diet. At the end of the probiotic feeding trial, fish were challenged with A. hydrophila, and the percentage of survival rates was recorded over 7 days. Results revealed that fish fed with A. veronii V03 demonstrated a significant improvement in growth and enhancement of innate immunity, including respiratory burst, myeloperoxidase, and lysozyme activities, and total immunoglobulin level compared with BD fed to fish. Relatively, expression of cytokines (MyD88, IL-1ß1, IL-8, and IL-10) and c- and g-type lysozymes were significantly up- and downregulated in lymphoid organs of fish. Moreover, dietary supplementation of A. veronii V03 exhibited significantly (p < 0.001) higher survival rates of DI (90%) and DII (96.66%) compared with BD (53.33%) fed fish against A. hydrophila infection. These findings help to understand the effects of probiotic A. veronii V03 administrated feed influences on growth and ailment resistance to A. hydrophila infection by regulating innate and systemic immunity in common carp fish.

Comparative genomic analysis of different virulence strains reveals reasons for the increased virulence of Aeromonas veronii.

Aeromonas veronii is an important zoonotic and aquatic agent. More and more cases have shown that it has caused huge economic losses in the aquaculture industry in addition to threatening human health. But the reasons for the increasing virulence of A. veronii are still unclear. In order to further understand the reasons for the increased virulence of A. veronii, we conducted a comparative analysis of the genomes of A. veronii with different virulence. The analysis revealed that there are multiple virulence factors, such as those related to fimbriae, flagella, toxins, iron ion uptake systems and type II, type III and type VI secretion systems in the virulent strain TH0426 genome. And comparative analysis showed that there were two complete type III secretion systems (API1 and API2), of which the API2 and iron ion transport system were unique to the TH0426 strain. In addition, TH0426 strain also has unique functional gene clusters, which may play important roles in terms of resisting infection, adapting to different environments and genetic evolution. These particular virulence factors and gene clusters may be the important reasons for the increased virulence. These insights will provide a reference for the study of the pathogenesis of A. veronii.

Genome sequencing and annotation of multi-virulent Aeromonas veronii XhG1.2 isolated from diseased Xiphophorus hellerii.

The present study was intended to elucidate the genomic basis of antibiotic resistance and hyper-virulence of the fish pathogen Aeromonas veronii XhG1.2 characterized in our previous work. The identity of XhG1.2 was confirmed through 16S rDNA sequence analysis and whole genome sequence analysis. The top-hit species distribution analysis of XhG1.2 sequence data revealed major hits against the Aeromonas veronii. The identification of virulence genes using the VFDB showed the genome of XhG1.2 to have the genes coding for the virulence factors viz. aerolysin, RtxA, T2SS, T3SS and T6SS. The presence of antibiotic resistance predicted through the CARD database analysis showed it to have the CephA3, OXA-12, adeF and pulvomycin resistance genes. By the phylogenetic and comparative genomic analysis, A. veronii species were found to have genes for toxin production. This also confirmed the pathogenicity and drug resistance of A. veronii XhG1.2 and also its potential to cause disease in diverse ornamental fishes.

Inactivation of the T6SS inner membrane protein DotU results in severe attenuation and decreased pathogenicity of Aeromonas veronii TH0426.

BACKGROUND: The inner membrane protein DotU of Aeromonas veronii is an important component of the minimal core conserved membrane proteome required for the formation of an envelope-transmembrane complex. This protein functions in a type VI secretion system (T6SS), and the role of this T6SS during the pathogenic process has not been clearly described. RESULTS: A recombinant A. veronii with a partial disruption of the dotU gene (720 bp of the in-frame sequence) (defined as ∆dotU) was constructed by two conjugate exchanges. We found that the mutant ∆dotU allele can be stably inherited for more than 50 generations. Inactivation of the A. veronii dotU gene resulted in no significant changes in growth or resistance to various environmental changes. However, compared with the wild-type strain colony, the mutant ∆dotU colony had a rough surface morphology. In addition, the biofilm formation ability of the mutant ∆dotU was significantly enhanced by 2.1-fold. Conversely, the deletion of the dotU gene resulted in a significant decrease in pathogenicity and infectivity compared to those of the A. veronii wild-type strain. CONCLUSIONS: Our findings indicated that the dotU gene was an essential participant in the pathogenicity and invasiveness of A. veronii TH0426, which provides a novel perspective on the pathogenesis of TH0426 and lays the foundation for discovering potential T6SS effectors.

Cadaverine reverse transporter (CadB protein) contributes to the virulence of Aeromonas veronii TH0426.

Aeromonas veronii is one of the main pathogens causing sepsis and ulcer syndrome in freshwater fish. Analysis of the results of epidemiological investigations in recent years has revealed that the virulence of A. veronii and its tolerance to drugs have been increasing year by year. Currently, most of the research on A. veronii focuses on its isolation, identification, and drug susceptibility, whereas research on its virulence factors and pathogenesis mechanisms is relatively rare. In this study, we identified and obtained the highly expressed TH0426 cadaverine reverse transporter (CadB) of A. veronii. We used efficient suicide plasmid-mediated homologous recombination to delete the cadB gene in TH0426 and constructed a cadB deletion strain. The LD50 of ΔcadB was 93.2 times higher than that of TH0426 in zebrafish, the toxicity of ΔcadB was 9.5 times less than that of TH0426 in EPC cells, and the biofilm formation ability of ΔcadB was 5.6-fold greater than that of TH0426. In addition, motility detection results indicated that ΔcadB had lost its swimming ability. The results of flagellar staining and TEM demonstrated that ΔcadB shed the flagella. In summary, the virulence and adhesion of A. veronii TH0426 were significantly decreased by the deletion of cadB, which might provide a theoretical basis for research into A. veronii virulence factors.

Functional analysis of hisJ in Aeromonas veronii reveals a key role in virulence.

Aeromonas veronii is an important aquatic zoonotic pathogen in humans and animals. In recent years, extracellular proteins from bacteria have been found to be the major pathogenic factors for aquatic animals. The aim of this study was to systematically analyze the extracellular proteins of nine sources of A. veronii and the effects of hisJ on virulence. We screened only the common proteins from nine different sources of A. veronii by liquid chromatography-tandem mass spectrometry and identified the gene hisJ. We then constructed ΔhisJ (deleted) and C-hisJ (complemented) variants of A. veronii TH0426 to assess the biological function of hisJ. While the ΔhisJ strain did not show altered growth (P > 0.05), we observed that it had reduced colony formation and biofilm formation and reduced adhesion to and invasion of epithelioma papulosum cyprini cells by 2.0-, 1.9-, and 10.8-fold, respectively. Additionally, infection experiments on zebrafish and mouse infection experiments showed that the virulence of the ΔhisJ strain was decreased by 865-fold (P < 0.001) compared with the wild-type strain; virulence of the complemented C-hisJ strain was reduced only 2.8-fold. Furthermore, in the context of hisJ deletion, flagella of A. veronii TH0426 were easily detached and the expression of virulence genes was downregulated. A persistence test (of bacterial colonies in crucian carp) showed that the number of bacteria in the immune organs of the ΔhisJ-infected group was lower than that in the wild-type-infected group. Overall, these results show that hisJ affects flagellar shedding, virulence, biofilm formation, adhesion, and invasion of A. veronii TH0426, and that hisJ is closely associated with virulence and plays a crucial role in its pathogenicity of A. veronii TH0426.

Virulence characteristics and antimicrobial resistance of Aeromonas veronii biovar sobria 312M, a clinical isolate.

Aeromonas are bacteria widely distributed in the environment, and some species are able to cause infections in humans, of which diarrhea is the most common. The objective of this study was to evaluate the presence of virulence and antimicrobial resistance associated characteristics in A. veronii biovar sobria strain 312M isolated from diarrheal stools. For this, the genome sequencing and phenotypical tests were performed. The draft genome annotation revealed several complete pathways associated with carbon metabolism and a mucin-desulfating sulfatase which may contribute to intestine colonization, and a large number of virulence-associated genes encoding structures associated with adhesion, toxins, and secretion systems. The strain exhibited swimming and swarming motility, biofilm formation, and hemolytic activity. It was resistant to ampicillin, ampicillin/sulbactam, and amoxicillin-clavulanic acid. Although a cphA gene encoding a narrow-spectrum carbapenase was identified in the strain genome, no carbapenemase activity was detected in the antimicrobial susceptibility test. When compared with other A. veronii with complete genomes, the main differences in virulence characteristics are related to lateral flagella and type III and VI secretion systems; the antimicrobial resistance spectrum also varied among strains. The results indicated that A. veronii biovar sobria 312M presents high virulence potential and resistance to limited classes of antimicrobials.

Functional Analysis of preA in Aeromonas veronii TH0426 Reveals a Key Role in the Regulation of Virulence and Resistance to Oxidative Stress.

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. This bacterium has caused serious economic losses in the aquaculture industry worldwide, and it has become an important zoonotic and aquatic agent. However, little is known about the molecular mechanism of pathogenesis of A. veronii. In this study, we first constructed an unmarked mutant strain (ΔpreA) by generating an in-frame deletion of the preA gene, which encodes a periplasmic binding protein, to investigate its role in A. veronii TH0426. Our results showed that the motility and biofilm formation ability of ΔpreA were similar to those of the wild-type strain. However, the adhesion and invasion ability in epithelioma papulosum cyprini (EPC) cells were significantly enhanced (2.0-fold). Furthermore, the median lethal dose (LD50) of ΔpreA was 7.6-fold higher than that of the wild-type strain, which illustrates that the virulence of the mutant was significantly enhanced. This finding is also supported by the cytotoxicity test results, which showed that the toxicity of ΔpreA to EPC cells was enhanced 1.3-fold relative to the wild type. Conversely, tolerance test results showed that oxidative stress resistance of ΔpreA decreased 5.9-fold compared to with the wild-type strain. The results suggest that preA may negatively regulate the virulence of A. veronii TH0426 through the regulation of resistance to oxidative stress. These insights will help to further elucidate the function of preA and understand the pathogenesis of A. veronii.

Oral Administration of Lactobacillus Casei Expressing Flagellin A Protein Confers Effective Protection against Aeromonas Veronii in Common Carp, Cyprinus Carpio.

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1ß, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.

Comparative genomics of Aeromonas veronii: Identification of a pathotype impacting aquaculture globally.

Aeromonas veronii is a gram-negative species abundant in aquatic environments that causes disease in humans as well as terrestrial and aquatic animals. In the current study, 41 publicly available A. veronii genomes were compared to investigate distribution of putative virulence genes, global dissemination of pathotypes, and potential mechanisms of virulence. The complete genome of A. veronii strain ML09-123 from an outbreak of motile aeromonas septicemia in farm-raised catfish in the southeastern United States was included. Dissemination of A. veronii strain types was discovered in dispersed geographical locations. Isolate ML09-123 is highly similar to Chinese isolate TH0426, suggesting the two strains have a common origin and may represent a pathotype impacting aquaculture in both countries. Virulence of strain ML09-123 in catfish in a dose-dependent manner was confirmed experimentally. Subsystem category disposition showed the majority of genomes exhibit similar distribution of genomic elements. The type I secretion system (T1SS), type II secretion system (T2SS), type 4 pilus (T4P), and flagellum core elements are conserved in all A. veronii genomes, whereas the type III secretion system (T3SS), type V secretion system (T5SS), type VI secretion system (T6SS), and tight adherence (TAD) system demonstrate variable dispersal. Distribution of mobile elements is dependent on host and geographic origin, suggesting this species has undergone considerable genetic exchange. The data presented here lends insight into the genomic variation of A. veronii and identifies a pathotype impacting aquaculture globally.

Role of Myeloperoxidase of northern snakehead (Channa argus) in Aeromonas veronii infection.

Myeloperoxidase (MPO) is a ferrous lysosomal protein with many immune functions that belongs to the heme peroxidase enzyme. In this study, the functions of MPO in the northern snakehead (Channa argus) were investigated by cloning an MPO cDNA sequence with a full length of 3181 bp. Homology analysis showed that northern snakehead MPO gene had the highest (81%) homology with mandarin fish (Siniperca chuatsi). In healthy northern snakehead, the MPO gene was expressed in the head-kidney, kidney, heart, gill, spleen, liver, and muscles but not midgut. After the northern snakehead was infected with Aeromonas veronii, the MPO gene expression varied in different tissues with low level in spleen, liver, gill and muscle, fluctuated in kidney and head-kidney and showed high level in heart. The result indicated that MPO might play an important role in the antimicrobial immune response of the northern snakehead.

Molecular characterization and interactome analysis of aerolysin (aer) gene from fish pathogen Aeromonas veronii: The pathogenicity inferred from sequence divergence and linked to histidine kinase (cheA).

Aerolysin (aer) is one of the most important and abundant virulence factors in the infection of fish by Aeromonas veronii. A comprehensive study on the molecular characterization and pathogenicity of the aer gene from 34 A. veronii isolates from diseased carp and catfish was carried out and its interactome was analysed to observe the functional correlations between aer and other proteins within the A. veronii network. The PCR-based amplification of aer from the 34 isolates of A. veronii showed more aer-positive isolates from catfish with a high pathogenic potential in the in vivo challenge test than the carp fish. The analysis of aer gene sequence from challenged fish revealed significant sequence divergence according to the types and geographical distribution of the fish. The networking analysis of aer from the model A. veronii B565 revealed histidine kinase (cheA) as the most functional interacting partner. The study of the interaction between aer from the experimental A. veronii and cheA demonstrated that the A chain of cheA plays a more important role than the corresponding B chain during contact, and a linker sequence of 15 residues controlled the entire interaction process. Therefore, cheA could be an excellent drug target for controlling A. veronii infection of fish.

Characterization, evolution, and expression analysis of TLR7 gene subfamily members in Mastacembelus armatus (Synbranchiformes: Mastacembelidae).

TLR7 subfamily members are important pattern recognition receptors participating in the recognition of pathogen-associated molecular patterns. In this study, we successfully identified 3 members of TLR7 subfamily from the spiny eel Mastacembelus armatus (MaTLR7, MaTLR8 and MaTLR9). The amino acid sequence identities of MaTLR7 and MaTLR8 with Monopterus albus TLR7 were 87.2% and 76.5%, respectively and the identity of MaTLR9 with Seriola lalandi TLR9 was 74.7%. The phylogenetic analysis revealed MaTLRs showed close relationship to other species in Synbranchiformes or Perciformes. Quantitative real-time PCR analysis revealed that they were expressed in all tested tissues and higher expression was found in spleen or gill. After infection with Aeromonas veronii, expression of MaTLR7, MaTLR8 and MaTLR9 were all significantly downregulated in spleen and kidney. Evolutionary analysis suggested that the ancestral lineages of teleost TLR8 and TLR9 had been subject to positive selection pressures and multiple Maximum likelihood methods recovered 3 positively selected sites in teleost TLR7, 4 in TLR8 and 8 in TLR9. Domain distribution revealed most positively selected sites were located in leucine-rich repeat domain. Our results will contribute to better understanding the antibacterial mechanism of TLRs and their co-evolution with pathogens.

Maltoporin (LamB protein) contributes to the virulence and adhesion of Aeromonas veronii TH0426.

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. More and more cases have shown that it has become an important zoonotic and aquatic agent. In this study, a A. veronii TH0426 mutant strain (ΔlamB) with an in-frame deletion removed nucleotides 10-1,296 of the lamB gene was firstly constructed to investigate its functions. The results showed that the LD50 value of the mutant ΔlamB to zebrafish and mice was 13.7-fold and 5.6-fold higher than those of the wild-type strain, respectively. The toxicity of wild-type strain to EPC cells was 2.1-fold and threefold higher than those of ∆lamB when infected for 1 and 2 hr. Furthermore, the ability of biofilm formation and the adhesion and invasion to EPC cells of ∆lamB significantly decreased for 5.6-fold and 1.8-fold separately. In addition, motility detection result indicated that ∆lamB lost the swimming ability. The results of flagellar staining and TEM demonstrated that the flagella of ∆lamB were shed. In general, the deletion of lamB gene caused a significant decrease in the virulence and adhesion of A. veronii TH0426, and it can be known that the lamB gene of A. veronii plays a crucial role in the pathogenesis.

Great effect of porin(aha) in bacterial adhesion and virulence regulation in Aeromonas veronii.

Aeromonas veronii is a serious pathogen which can infect mammals and aquatic organisms and causes irreparable damage to fish aquaculture. It has been demonstrated that adhesion to host surface and cells is the initial step in bacterial pathogenesis. Previous study found that bacterial weaken motility probably caused by the absence of flagellar related genes. In this study, we generated the aha deletion and complementary strains and found that two strains can be stably inherited for more than 50 generations. No significant change was found in the growth of mutant △aha. But the ability of biofilm formation, the adhesion and invasion to EPC cells significantly decreased for 3.7-fold and 2.3-fold respectively. Due to aha gene deletion, the stability of A. veronii flagellar was severely declined and the mutant △aha with no mobility. Compared with the wild-type TH0426, the pathogenicity of A. veroniiaha-deleted strain to zebrafish and mice reduced significantly and virulence attenuated severely. Cytotoxicity experiment also proved that mutant △aha showed much weaker virulence at the same time infection. The consequences declared that the stability of flagellar decreased severely with porin missing and lost the motility. Porin regulated by aha gene is essential for the adhesion and virulence of A. veronii. Thence, the mutant △aha of A. veronii provides an important tool for further concentration on the pathogenic mechanism of A. veronii.