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The TonB system is required for the active transport of iron compounds across the outer membrane in Gram-negative bacteria. Our previous data indicated that three TonB systems act coordinately to contribute to the motility of Aeromonas hydrophila NJ-35. In this study, we found that flagellum biogenesis was defective in the ΔtonB123 mutant. Subcellular localization indicated that the flagellin subunits FlaA and FlaB were trapped in the cytoplasm of ΔtonB123 mutant with reduced molecular mass. Overexpression of FlaA or FlaB in the ΔtonB123 mutant was unable to restore the secretion of flagellin subunits. Further investigation demonstrated that flagellins in the ΔtonB123 mutant showed a weak affinity for the flagellin-specific chaperone FliS, which is necessary for the export of flagellins. Deglycosylation analysis indicated that flagellins in the cytoplasm of the ΔtonB123 mutant were almost nonglycosylated. Our data suggested that disruption of tonB123 impairs the formation of flagella by inhibiting flagellin glycosylation and decreasing the binding affinity of flagellin for the chaperone FliS. Taken together, our findings indicate a new role of the TonB system in flagellar biogenesis in A. hydrophila., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA 2 B 2 , one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA 2 B 2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA 2 B 2 pump was completely abrogated by tonB123 deletion. In addition, macA 2 B 2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA 2 B 2 -mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA 2 B 2 -mediated macrolide efflux.

Background: Aeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections., Results: Five phages against pathogenic A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues., Conclusions: Five A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy.

The type VI secretion system (T6SS) is a multiprotein weapon that kills eukaryotic predators or prokaryotic competitors by delivering toxic effectors. Despite the importance of T6SS in bacterial environmental adaptation, it is still challenging to systematically identify T6SS effectors because of their high diversity and lack of conserved domains. In this report, we discovered a putative effector gene, U876-17730, in the whole genome of Aeromonas hydrophila NJ-35 based on the reported conservative domain DUF4123 (domain of unknown function), with two cognate immunity proteins encoded downstream. Phylogenetic tree analysis of amino acids indicates that AH17730 belongs to the Tle1 (type VI lipase effector) family, and therefore was named Tle1 AH . The deletion of tle1 AH resulted in significantly decreased biofilm formation, antibacterial competition ability and virulence in zebrafish (Danio rerio) when compared to the wild-type strain. Only when the two immunity proteins coexist can bacteria protect themselves from the toxicity of Tle1 AH . Further study shows that Tle1 AH is a kind of phospholipase that possesses a conserved lipase motif, Gly-X-Ser-X-Gly (X is for any amino acid). Tle1 AH is secreted by T6SS, and this secretion requires its interaction with an associated VgrG (valine-glycine repeat protein G). In conclusion, we identified a T6SS effector-immunity pair and verified its function, which lays the foundation for future research on the role of T6SS in the pathogenic mechanism of A. hydrophila.

Aeromonas hydrophila is the causative agent of motile Aeromonad septicemia in fish. Previous studies have shown that the myo-inositol metabolism is essential for the virulence of this bacterium. IolR is a transcription inhibitor that negatively regulates myo-inositol metabolic activity. While in the process of studying the inositol catabolism in A. hydrophila Chinese epidemic strain NJ-35, we incidentally found that ΔiolR mutant exhibited obvious autoaggregation and increased biofilm formation compared to the wild type. The role of surface proteins in A. hydrophila autoaggregation was confirmed by different degradation treatments. Furthermore, calcium promotes the formation of aggregates, which disappear in the presence of the calcium chelator EGTA. Transcriptome analysis, followed by targeted gene deletion, demonstrated that biofilm formation and autoaggregation caused by the inactivation of iolR was due to the increased transcription of a RTX-family adhesion gene, rmpA. Further, IolR was determined to directly regulate the transcription of rmpA. These results indicated that iolR is negatively involved in autoaggregation and biofilm formation in A. hydrophila, and this involvement was associated with its inhibition on the expression of rmpA.

NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.

The TonB system functions in iron transport and has been identified in certain Gram-negative bacteria. Recently, we reported three TonB systems in the Aeromonas hydrophila Chinese epidemic strain NJ-35, but the functions of these systems have not been thoroughly elucidated to date. In this study, we investigated the role of these TonB systems in A. hydrophila iron utilization and virulence. We found that tonB1 and tonB2 were preferentially transcribed in iron-chelated conditions, where gene expression levels were approximately 8- and 68-fold higher compared with iron-rich conditions, respectively; tonB3 was consistently transcribed at a low level under iron-repleted and iron-depleted conditions. Only the TonB2 system was required to utilize iron-binding proteins. The tonB123 mutant showed increased susceptibility to erythromycin and roxithromycin. In addition, all three tonB genes were involved in A. hydrophila virulence in zebrafish, and various phenotypes associated with environmental survival were changed with varying degrees in each tonB mutant. TonB2 plays a relatively major role in adhesion, motility, and biofilm formation, while TonB3 is more involved in the anti-phagocytosis of A. hydrophila. In each observed phenotype, no significant difference was found between the single- and double-deletion mutants, whereas the triple-deletion mutant exhibited the most serious defects, indicating that all three TonB systems of A. hydrophila coordinately complement one another. In conclusion, this study elucidates the importance of TonB in iron acquisition and virulence of A. hydrophila, which lays the foundation for future studies regarding the survival mechanisms of this bacterium in iron-restricted environments.

This study explored the immunomodulatory effect and inhibition effects of the candidate probiotic Lactococcus lactis 16-7, which was isolated from crucian carp, on Aeromonas hydrophila infection in crucian carp. The experimental fish were divided into two groups; one was fed a diet supplemented with L. lactis, while the other was fed the control probiotic-free diet. After feeding for 42 d with the experimental diets, the fish that received the diet supplemented with probiotics exhibited a significantly enhanced serum superoxide dismutase activity, phagocytic activities of innate immune cells, and the expression levels of immune-related genes [interferon-γ (INF-γ), interleukin-1β (IL-1β), interleukin-11 (IL-11), tumour necrosis factor α (TNF-α) and myeloid differentiation factor 88 (MyD88)], indicating that L. lactis 16-7 could activate the non-specific immune system of crucian carp. At the end of the feeding trial, the crucian carps in each group were orally infected with A. hydrophila NJ-35. The results show that L. lactis 16-7 could prevent the increase in d-lactic acid concentration and inflammatory response caused by A. hydrophila in crucian carp. Compared with A. hydrophila group, L. lactis 16-7 preserved the integrity of intestinal villi and mitigated A. hydrophila-induced reduce in the transcriptional levels of tight junction (TJ) proteins zonula occludens-1 (ZO-1) and occludin, indicating that L. lactis 16-7 could reduce intestinal mucosal barrier damage and inflammation induced by A. hydrophila in crucian carp. In addition, L. lactis 16-7 could effectively antagonize the colonization of A. hydrophila in the intestine. Overall, these data clearly indicate that L. lactis 16-7 has the potential to be developed as a probiotic agent against A. hydrophila infection in aquaculture., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Aeromonas hydrophila is a potential zoonotic pathogen and primary fish pathogen. With overlapping characteristics, multiple isolates are often mislabelled and misclassified. Moreover, the potential pathogenic factors among the publicly available genomes in A. hydrophila strains of different origins have not yet been investigated., Results: To identify the valid strains of A. hydrophila and their pathogenic factors, we performed a pan-genomic study. It revealed that there were 13 mislabelled strains and 49 valid strains that were further verified by Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and in silico multiple locus strain typing (MLST). Multiple numbers of phages were detected among the strains and among them Aeromonas phi 018 was frequently present. The diversity in type III secretion system (T3SS) and conservation of type II and type VI secretion systems (T2SS and T6SS, respectively) among all the strains are important to study for designing future strategies. The most prevalent antibiotic resistances were found to be beta-lactamase, polymyxin and colistin resistances. The comparative analyses of sequence type (ST) 251 and other ST groups revealed that there were higher numbers of virulence factors in ST-251 than in other STs group., Conclusion: Publicly available genomes have 13 mislabelled organisms, and there are only 49 valid A. hydrophila strains. This valid pan-genome identifies multiple prophages that can be further utilized. Different A. hydrophila strains harbour multiple virulence factors and antibiotic resistance genes. Identification of such factors is important for designing future treatment regimes.

Aeromonas hydrophila is an important aquatic microorganism that can cause fish hemorrhagic septicemia. In this study, we identified a novel LysR family transcriptional regulator (LahS) in the A. hydrophila Chinese epidemic strain NJ-35 from a library of 947 mutant strains. The deletion of lahS caused bacteria to exhibit significantly decreased hemolytic activity, motility, biofilm formation, protease production, and anti-bacterial competition ability when compared to the wild-type strain. In addition, the determination of the fifty percent lethal dose (LD 50 ) in zebrafish demonstrated that the lahS deletion mutant ( ΔlahS ) was highly attenuated in virulence, with an approximately 200-fold increase in LD 50 observed as compared with that of the wild-type strain. However, the ΔlahS strain exhibited significantly increased antioxidant activity (six-fold). Label-free quantitative proteome analysis resulted in the identification of 34 differentially expressed proteins in the ΔlahS strain. The differentially expressed proteins were involved in flagellum assembly, metabolism, redox reactions, and cell density induction. The data indicated that LahS might act as a global regulator to directly or indirectly regulate various biological processes in A. hydrophila NJ-35, contributing to a greater understanding the pathogenic mechanisms of A. hydrophila.

The type VI secretion system (T6SS) has been considered as a crucial factor in bacterial competition and virulence. The hemolysin co-regulated protein (Hcp) is the hallmark of T6SS. The secretion of Hcp in Aeromonas hydrophila Chinese epidemic strain NJ-35 indicated a functional T6SS. In this study, three copies of the hcp gene were identified in the genome of strain NJ-35. We targeted these Hcp family proteins for generating deletion mutants. These mutants showed varying levels in Hcp production, the interaction with other bacteria or eukaryotic cells, and bacterial virulence. Hcp1 was necessary for T6SS assembly and played a predominant role in the bacterial competition; Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A. hydrophila virulence in zebrafish and survival against the predation of Tetrahymena, and Hcp3 positively influenced the biofilm formation and bacterial adhesion. These findings illustrate that the T6SS of A. hydrophila NJ-35 is active, and the three Hcp family proteins take part in different processes in environmental adaptation and virulence of this bacterium. This study will provide valuable insights into our understanding of microbial interactions and thus contribute to a broader effort to manipulate these interactions for therapeutic or environmental benefit.

Persistence of Aeromonas hydrophila in aquatic environments is the principle cause of fish hemorrhagic septicemia. Protistan predation has been considered to be a strong driving force for the evolution of bacterial defense strategies. In this study, we investigated the adaptive traits of A. hydrophila NJ-35, a carp pathogenic strain, in response to Tetrahymena thermophila predation. After subculturing with Tetrahymena , over 70% of A. hydrophila colonies were small colony variants (SCVs). The SCVs displayed enhanced biofilm formation, adhesion, fitness, and resistance to bacteriophage infection and oxidative stress as compared to the non- Tetrahymena- exposed strains. In contrast, the SCVs exhibited decreased intracellular bacterial number in RAW264.7 macrophages and were highly attenuated for virulence in zebrafish. Considering the outer membrane proteins (OMPs) are directly involved in bacterial interaction with the external surroundings, we investigated the roles of OMPs in the antipredator fitness behaviors of A. hydrophila . A total of 38 differentially expressed proteins were identified in the SCVs by quantitative proteomics. Among them, three lipoproteins including SurA, Slp, and LpoB, and a serine/threonine protein kinase (Stpk) were evidenced to be associated with environmental adaptation of the SCVs. Also, the three lipoproteins were involved in attenuated virulence of SCVs through the proinflammatory immune response mediated by TLR2. This study provides an important contribution to the understanding of the defensive traits of A. hydrophila against protistan predators.

Outbreaks of motile Aeromonad septicemia (MAS) in fish caused by sequence type (ST) 251 Aeromonas hydrophila have become a prominent problem for the aquaculture industry. The pathogenesis of A. hydrophila is very complicated, and some virulence factors remain to be identified. In this study, to identify novel virulence-related factors, ST251 A. hydrophila strain NJ-35 was used as the parental strain to construct a mutant library comprising 1030 mutant strains by transposon insertion mutagenesis. Subsequently, 33 virulence-attenuated transposon insertion mutants were identified using Tetrahymena and zebrafish as model hosts in sequence. Thermal asymmetric interlaced (Tail)-PCR and Southern blot analysis identified 21 single transposon insertion sites. Seven of the insertion sites are located in non-coding regions, whereas the other 14 insertion sites are located in genes, including aroA, rmlA, rtxA, chiA and plc. All insertion mutants exhibited attenuated virulence in Tetrahymena and zebrafish. Furthermore, the relationship of two genes, chiA and trkH, to virulence was confirmed by gene inactivation and subsequent restoration assays. This study provides new information about the genetic determinants of A. hydrophila pathogenicity and validates the Aeromonas-Tetrahymena co-culture model for high-throughput screening of A. hydrophila virulence factors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of Aeromonas hydrophila when co-cultured with the bacteriovorous ciliate Tetrahymena thermophila and investigated bacterial gene expression associated with the co-culture. Virulent A. hydrophila strains were found to have ability to evade digestion in the vacuoles of this protozoan. In A. hydrophila , a total of 116 genes were identified as up-regulated following co-culture with T. thermophila by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, msrA , and msrB , in A. hydrophila . Compared to the wild-type, the mutants Δ msrA and Δ msrAB displayed significantly reduced resistance to predation by T. thermophila , and 50% lethal dose (LD 50 ) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses.

The growth-stimulating effects of catecholamine stress hormones have been demonstrated in many pathogens. However, catecholamine-induced growth and its underlying mechanisms remain poorly understood in Aeromonas hydrophila . The present study sought to demonstrate that norepinephrine (NE), epinephrine (Epi), dopamine (Dopa), and L-dopa stimulate the growth of A. hydrophila in iron-restricted media containing serum. NE exhibited the strongest growth stimulation, which could be blocked by adrenergic antagonists. Furthermore, it was demonstrated that NE could sequester iron from transferrin, thereby providing a more accessible iron source for utilization by A. hydrophila . The deletion of the amoA gene associated with amonabactin synthesis revealed that the amonabactin siderophore is not required for NE-stimulated growth. However, the deletion of the TonB2 energy transduction system resulted in the loss of growth promotion by NE, indicating that a specific TonB-dependent outer membrane receptor might be involved in the transport of iron from transferrin. Collectively, our data show that catecholamine sensing promotes the growth of A. hydrophila in a manner that is dependent on the TonB2 energy transduction system.

Motile aeromonad septicemia (MAS) caused by Aeromonas hydrophila is one of the common bacterial causes of fish mortalities. Prophylactic vaccination against this and other diseases is essential for continued growth of aquaculture. The type VI secretion system (T6SS) plays a crucial role in the virulence of A. hydrophila. The hemolysin co-regulated protein (Hcp) is an integral component of the T6SS apparatus and is considered a hallmark of T6SS function. Here, the T6SS effector Hcp was expressed and characterized, and its immunogenicity and protective efficacy were evaluated in common carp (Cyprinus carpio). Hcp secretion was found to be strongly induced by low temperature in A. hydrophila. Immunoblot analysis demonstrated that Hcp is conserved among A. hydrophila strains of different origins. The vaccination with recombinant Hcp resulted in an increased survival (46.67%) in common carp during a 10-day challenge time compared to non-vaccinated fish (7.14%). The vaccinated fish also showed the significantly increased levels of IgM antibody in serum and cytokines such as inerleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in kidney, spleen and gills. The recombinant Hcp shows promise as a vaccine candidate against A. hydrophila., (Copyright © 2015. Published by Elsevier Ltd.)

Outbreaks in fish of motile Aeromonad septicemia (MAS) caused by Aeromonas hydrophila have caused a great concern worldwide. Here, for the first time, we provide two complete genomes of epidemic A. hydrophila strains isolated in China. To gain an insight into the pathogenicity of epidemic A. hydrophila, we performed comparative genomic analyses of five epidemic strains belonging to sequence type (ST) 251, together with the environmental strain ATCC 7966(T). We found that the known virulence factors, including a type III secretion system, a type VI secretion system and lateral flagella, are not required for the high virulence of the ST251 clonal group. Additionally, our work identifies three utilization pathways for myo-inositol, sialic acid and L-fucose providing clues regarding the factors that underlie the epidemic and virulent nature of ST251 A. hydrophila. Based on the geographical distribution and biological resources of the ST251 clonal group, we conclude that ST251 is a high-risk clonal group of A. hydrophila which may be responsible for the MAS outbreaks in China and the southeastern United States.

Aeromonas hydrophila is a fish pathogen causing systemic infections in aquatic environments, and determining its antigenic proteins is important for vaccine development to reduce economic losses in aquaculture worldwide. Here, an immunoproteomic approach was used to identify immunogenic outer membrane proteins (OMPs) of the Chinese vaccine strain J-1 using convalescent sera from Chinese breams. Seven unique immunogenic proteins were identified by two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF-MS). One protein of interest, Omp38, was expressed, and its immunogenicity and protective efficacy were evaluated in Chinese breams. The two groups of fish immunized with the inactivated vaccine and recombinant Omp38 protein showed significant serum IgM antibody levels after vaccination, compared with the fish injected with PBS buffer. In addition, the superoxide dismutase (SOD) activity, lysozyme (LSZ) activity and phagocytosis activity of head kidney lymphocytes of immunized groups were significantly higher than those of the control. The fish receiving inactivated vaccine and recombinant Omp38 protein developed a protective response to a live A. hydrophila challenge 45 days post-immunization, as demonstrated by increased survival of vaccinated fish over the control and by decreased histological alterations in vaccinated fish. Furthermore, protective effect was better in Omp38 group than in the inactivated vaccine group. These results suggest that the recombinant Omp38 protein could effectively stimulate both specific and non-specific immune responses and protect against A. hydrophila infection. Therefore, Omp38 may be developed as a potential vaccine candidate against A. hydrophila infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: The enzyme elastase is an important virulent factor of the opportunistic pathogen Aeromonas hydrophila. To know better about the molecular characterization of this enzyme, we purified the elatase and investigated its property and activity., Methods: We cloned a structural gene ahyB encoding for the extracellular elastase of A. hydrophila J-1 and expressed it in E. coli BL21 by using pET-32a as vector. We purified the recombinant enzyme using His Bind Resin Purification Kit and recovered the elastolytic activity of the purified protein by incubation in a buffer containing 6 mol/L guanidine HCl and subsequent removal of denaturant by dilution. In addition, we also purified the native enzyme from the culture supernatant of A. hydrophila J-1 by 30 to 60% ammonium sulfate fractionation, anion exchange chromatography and sephaceryl chromatography. We compared the properties of the two elastase preparations., Results: Native enzyme showed a pH optimum at 8.5, but recombinant enzyme at 10.0. Compared with the native enzyme, the recombinant enzyme was more stable for heat. Both the elastase preparations showed some identical properties concerning inhibitors,which were both mainly inhibited by EDTA, the cation chelator, and OPA, a Zn2+ /Fe2+ protease inhibitor. However,the recombinant elastase had higher tolerance than native enzyme against the inhibitors. And the metal cation Zn2+ and Fe2+ strongly inhibited the enzyme activity., Conclusion: The recombinant product showed the similar enzymatic characteristics as the native enzyme from A. hydrophila J-1 and the two elastases belonged to the zinc-and-iron-dependant metalloendopeptidase. This is the first report on the recombinant expression and subsequent molecular chracterization analysis of A. hydrophila elastase.

The bacterial ghost (BG) system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria, and can induce humoral and cellular immune response, including mucosal immune responses. Plasmid pElysis consisting E gene was transformed into AhJ-1. Through shifting the culture temperature from 28 degrees C to 42 degrees C, A. hydrophila J-1 (pElysis) was induced to lyse and the OD600 value of culture media was measured every 15 minutes during the induction. The lysed bacteria were observed by scanning electron microscopy (SEM). The A. hydrophila ghosts (AHG) used as oral vaccine were also investigated. The OD600 value of A. hydrophila J-1(pElysis) began to decline after 30 min of induction, and after 75 min of induction, the OD600 value decline speed become slowly. The efficiency of ghost induction in non-lyophilized A. hydrophila was 99.99%, 16 hours post induced, no live bacteria can be detected in culture. Scanning electron microscopy observation proved that most lysed bacteria were emptied. Fish vaccination experiments shows that the antibody evoked highest degree after 5 weeks by oral administration of bacterial ghost vaccine and the agglutination antibody titer reached 2(7) and continued two weeks, while the agglutination antibody titer of formalin killed vaccine only reached 26 and only maintained one week. After challenged with the parent strain J-1, the survival rate of bacterial ghost vaccinated fish was higher than the control group and formalin killed vaccine group, the relative percent survival (RPS) was 78.95% (16/20), but the RPS of formalin killed vaccine group was 57.9% (12/20). This suggests that the bacterial ghost vaccine has higher potential to induce protective adaptive immunity than normal vaccine.

Invasion of Aeromonas hydrophila strain Ah J-1 isolated from diseased fish to cultured HEp-2cells monolayer was evaluated by the recovery of gentamincin-resistant (Gm') bacteria from Triton X-100 cell lysates. The invasive efficiency (IE) could reach to 0.2% at 37 degrees C. In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors. Bacterial internalization of Ah J-1 involved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization. Staurosporine (a protein kinase C inhibitor) and Sodium orthovanadate (a protein tyrosine phosphatase inhibitor) accelerated internalization of Ah J-1 into HEp-2 cells.

Aeromonas hydrophila (Ah) biofilm (BF) model in vitro was established and antibiotic effects of 11 antimicrobials on BF and free-cell(FC) bacteria were tested. After the Ah J-1 on the surfaces of the silicon pieces was incubated for 7 days in TSB, the intact BF was formed by detection of AgNO3 solution staining. The FC bacteria were resistant to Penicillin but susceptible to Enrofloxacin and Fulgram. Their minimum bactericidal concentrations (MBCs) were 256 microg/mL, 0.03 microg/mL and 0.25 microg/mL respectively. Florfenicol had a strong antibacterial effect on BF bacteria and the ratio of MBC between BF and FC bacteria was 2 to 1, but more than 32 to 1 occurred for Kanamycin, Penicillin and Neomycin. Morphology and structure of BFs with/without the treatment of Enrofloxacin were observed under scanning electron microscope, while the bactericidal curve was detected. Enrofloxacin could eradicate the FC bacteria completely but not the BF bacteria at a concentration of 4 fold MBC. The 32 fold MBC Enrofloxacin could entirely kill the FC bacteria in 4h, but in 24h for BF bacteria. The result suggested that BF enabled Ah to have strong resistance to antimicrobials and the potential influence of BF should be highly considered.

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux. [ABSTRACT FROM AUTHOR]