Microbial influence on the larval survival of Japanese eel Anguilla japonica: Antibiotic-mediated alterations and biomarker isolation.

In rearing systems for the Japanese eel Anguilla japonica, although it is assumed that microorganisms influence larval survival and mortality, particularly during the early stages of growth, the effects of bacterial communities on larval survival have yet to be sufficiently determined. In this study, we compared the bacterial communities associated with larval survival at three stages of eel growth. To artificially alter bacterial communities and assess larval survival, eel larvae were treated with 11 types of antibiotic, and larval survival and bacterial characteristics were compared between the antibiotic-treated and antibiotic-free control groups. Throughout the three growth stages, eels treated with four antibiotics (polymyxin B, tetracycline, novobiocin, and erythromycin) had survival rates higher than those in the control groups. The bacterial communities of surviving larvae in the control and antibiotic groups and dead larvae in the control groups were subsequently analyzed using 16S rRNA gene amplicon sequencing. PERMANOVA analysis indicated that these three larval groups were characterized by significantly different bacterial communities. We identified 14 biomarker amplicon sequence variants (ASVs) of bacterial genera such as Oceanobacter, Alcanivorax, Marinobacter, Roseibium, and Sneathiella that were enriched in surviving larvae in the antibiotic treatment groups. In contrast, all four biomarker ASVs enriched in dead larvae of the control groups were from bacteria in the genus Vibrio. Moreover, 52 bacterial strains corresponding to nine biomarkers were isolated using a culture method. To the best of our knowledge, this is the first study to evaluate the bacterial communities associated with the survival and mortality of larvae in during the early stages of Japanese eel growth and to isolate biomarker bacterial strains. These findings will provide valuable insights for enhancing larval survival in the eel larval rearing systems from a microbiological perspective.

Transcriptome Analysis of Host Anti-Vibrio harveyi Infection Revealed the Pathogenicity of V. harveyi to American Eel (Anguilla rostrata).

Vibrio harveyi, a recently discovered pathogenic bacterium isolated from American eels (Anguilla rostrata), poses uncertainties regarding its pathogenesis in American eel and the molecular mechanisms underlying host defense against V. harveyi infection. This study aimed to determine the LD50 of V. harveyi in American eel and assess the bacterial load in the liver, spleen, and kidney post-infection with the LD50 dose. The results showed that the LD50 of V. harveyi via intraperitoneal injection in American eels over a 14d period was determined to be 1.24 × 103 cfu/g body weight (6.2 × 104 cfu/fish). The peak bacterial load occurred at 36 h post-infection (hpi) in all three organs examined. Histopathology analysis revealed hepatic vein congestion and thrombi, tubular vacuolar degeneration, and splenic bleeding. Moreover, quantitative reverse transcription polymerase chain reaction (qRT-PCR) results indicated significant up or downregulation of 18 host immune- or anti-infection-related genes post 12 to 60 hpi following the infection. Additionally, RNA sequencing (RNA-seq) unveiled 7 hub differentially expressed genes (DEGs) and 11 encoded proteins play crucial roles in the anti-V. harveyi response in American eels. This study firstly represents the comprehensive report on the pathogenicity of V. harveyi to American eels and RNA-seq of host's response to V. harveyi infection. These findings provide valuable insights into V. harveyi pathogenesis and the strategies employed by the host's immune system at the transcriptomic level to combat V. harveyi infection.

Comparative phenotype and transcriptome analysis revealed the role of ferric uptake regulator (Fur) in the virulence of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata).

Vibrio harveyi is commonly found in salt and brackish water and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δfur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δfur mutant exhibited weaker biofilm formation and swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels; compared to the wild-type strain, the mutant strain showed time and diameter differences in growth and haemolysis, respectively. Additionally, the adhesion ability of the mutant strain was significantly decreased. Moreover, there were 15 different biochemical indicators observed between the two strains. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δfur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II and type VI secretion systems (T2SS and T6SS), amino acid synthesis and transport and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δfur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δfur strain. These findings suggest that the virulence of the Δfur strain was significantly decreased, which is closely related to phenotype changing and gene transcript regulation.

RNA-seq analysis revealed the pathogenicity of Vibrio vulnificus to American eel (Anguilla rostrata) and the strategy of host anti-V. vulnificus infection.

Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.

Anguilla sp. diseases diagnoses and treatments: The ideal methods at the crossroads of conservation and aquaculture purposes.

Anguilla anguilla, A. japonica and A. rostrata are the most fished and consumed eel species. However, these species are Critically Endangered, Endangered and Endangered, respectively. A combination of factors is thought to be responsible for their decline including fisheries, climate change, habitat destruction, barriers to migration, pollution and pathogens. Among them, viruses, bacteria and parasites are causing weakening of wild eels and serious economic losses for fishermen and eel farmers. Early detection of pathogens is essential to provide appropriate responses both for conservation reasons and to limit economic losses. Classic diagnosis approaches are time consuming and invasive and usual treatments, for example, antipathogenic substances are becoming obsolete because of pathogen resistance and environmental impact problems. The need for early and non-invasive diagnostic methods as well as effective and environmentally friendly treatments has increased. Vaccine development and diet supplementation have known a growing interest since their use could allow prevention of diseases. In this review, we summarize the main pathogens-viruses, bacteria and parasites-of the three northern temperate eel species, the methods used to detect these pathogens and the different treatments used. We discussed and highlighted the need for non-invasive, rapid and efficient detection methods, as well as effective and environmentally friendly treatments for both conservation and aquaculture purposes.

Comparative transcriptome and phenotype analysis revealed the role and mechanism of ompR in the virulence of fish pathogenic Aeromonas hydrophila.

Aeromonas hydrophila B11 strain was isolated from diseased Anguilla japonica, which had caused severe gill ulcers in farmed eel, causing huge economic losses. EnvZ-OmpR is a model two-component system in the bacteria and is widely used in the research of signal transduction and gene transcription regulation. In this study, the ompR of A. hydrophila B11 strain was first silenced by RNAi technology. The role of ompR in the pathogenicity of A. hydrophila B11 was investigated by analyzing both the bacterial comparative transcriptome and phenotype. The qRT-PCR results showed that the expression of ompR in the ompR-RNAi strain decreased by 97% compared with the wild-type strain. The virulence test showed that after inhibition of the ompR expression, the LD50 of A. hydrophila B11 decreased by an order of magnitude, suggesting that ompR is involved in the regulation of bacterial virulence. Comparative transcriptome analysis showed that the expression of ompR can directly regulate the expression of several important virulence-related genes, such as the bacterial type II secretion system; moreover, ompR expression also regulates the expression of multiple genes related to bacterial chemotaxis, motility, adhesion, and biofilm formation. Further studies on the phenotype of A. hydrophila B11 and ompR-RNAi also confirmed that the downregulation of ompR expression can decrease bacterial chemotaxis, adhesion, and biofilm formation.

Immunization of a novel bivalent outer membrane protein simultaneously resisting Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, European eels (Anguilla anguilla) were immunized by the bivalent expression products of the outer membrane protein (Omp) gene from A. hydrophila (OmpⅡ) and E. anguillarum (OmpA), and the effects of the bivalent protein (rOmpⅡ-A) on the immune function of the European eel were detected. Three hundred eels were divided average into three groups of PBS, adjuvant and rOmp. Eels of three goups were injected intraperitoneal with 0.2 mL of PBS (0.01 mol/L, pH7.4), PBS + F (PBS mixed equal volume of freund's uncomplete adjuvant) or rOmpⅡ-A (1 mg mL-1 rOmpⅡ-A mixed equal volume of freund's uncomplete adjuvant). Four immune-related genes expression, proliferation of whole blood cells, serum and skin mucus antibody titer, superoxide dismutase (SOD) activity and the relative percent of survival (RPS) were studied at different days (or hours) post the immunization. The results showed that the igm, lysC, mhc2 and sod gene in the liver, spleen, kidney and intestine tract were significant increased in the Omp group; On the 28 day post the immunization (dpi), blood cell proliferation was increased in the Omp group, and on the 14, 21, 28 and 42 dpi, antibody titers in serum and mucus of the Omp group were significantly higher than that of the PBS and adjuvant group, regardless of coating with bacteria or Omp antigen. The SOD activity of Omp group increased significantly in liver, kidney, skin mucus and serum from 14 to 42 dpi, especially in serum. Eels chanllenged by A. hydrophila, E. anguillarum and V. vulnificus in the bivalent Omp group showed the RPS were 83.33%, 55.56% and 44.44%, respectively. The results of this study showed that immunization of the bivalent Omp could effectively improve the immune function of European eels, and produced effectively protection to A. hydrophila and E. anguillarum infection. Simultaneously, the bivalent Omp also produced distinct cross-protection to the eels challenged by V. vulnificus.

Chitosan and Oregano Oil Treatments, Individually or in Combination, Used To Increase the Shelf Life of Vacuum-Packaged, Refrigerated European Eel (Anguilla anguilla) Fillets.

We investigated the impact of chitosan and oregano essential oil (EO) individually or in combination on the quality of eel fillets in vacuum packaging (VP) and stored under refrigeration (4°C). Treatments studied were (i) control eel fillets stored in VP (E), (ii) eel fillets treated with 0.3% (v/w) oregano EO and stored in VP (E-OR), (iii) eel fillets treated with 2.0% (w/v) chitosan and stored in VP (E-CH), and (iv) eel fillets treated with 2.0% (w/v) chitosan and 0.3% (v/w) oregano EO and stored in VP (E-CH-OR). Treatments E-CH-OR and E-CH significantly reduced counts of mesophilic bacteria, Pseudomonas, Shewanella, and yeasts and molds during storage. Use of chitosan alone or in combination with oregano EO led to a significant reduction in concentrations of trimethylamine nitrogen and total volatile basic nitrogen in fillets, which led to lower concentrations of thiobarbituric acid reactive substances compared with the control samples. The eel samples in the E-CH and E-CH-OR groups were sensorially acceptable during the entire refrigerated storage period of 18 days. Presence of chitosan in the E-CH and E-CH-OR fillets did not negatively affect the taste of the fillets. E-CH fillets received a higher taste score than did E-CH-OR fillets probably because of the distinct and "spicy" lemon taste of chitosan, which was well received by the sensory panel. Based on overall sensory data (based on mean sensory scores of odor and taste), the shelf life was 6 days for the control fillets, 10 days for the E-OR fillets, and >18 days for the E-CH and E-CH-OR fillets stored in VP at 4°C. Overall, chitosan-treated eel fillets had lower microbial loads and a longer shelf life compared with the controls. Chitosan-treated eel fillets were preferred over oregano-treated fillets. Chitosan alone or in combination with oregano could be used as a preservative treatment and shelf-life extender for other seafoods.

Immunogenicity of a bivalent protein as a vaccine against Edwardsiella anguillarum and Vibrio vulnificus in Japanese eel (Anguilla japonica).

The OMPs A (OmpA)-of Edwardsiella anguillarum and OmpU of V. vulnificus have been proven to be good antigens. In this study, after construction of a vector, a new recombinant Omp (rOMP) containing both OmpA and OmpU was expressed and purified. Then, the Japanese eels (Anguilla japonica) were intraperitoneally (i.p.) injected with the phosphate-buffered saline (PBS group), formalin-killed-cell (FKC group) or the recombinant Omp (rOMP group). The stimulation index of the whole blood cells in eels from FKC group was significantly higher than the eels from PBS and rOMP groups at 28 dpi; serum titers of anti-E. anguillarum and anti-V. vulnificus antibody of eels from FKC and rOMP group increased significantly at 21 and 28 dpi; in the rOMP group, eels serum titer stayed at a high level on 42 dpi. The activities of lysozyme in skin mucus, liver, kidney, and serum in three groups exhibited considerable changes. The relative percent survival (RPS) rate of eels from rOMP group were 100% and 83% when challenged with V. vulnificus or E. anguillarum. These results indicated that inoculation of rOMP would protect Japanese eels against the infection by E. anguillarum and V. vulnificus.

Influence of bacteriophages cocktail on European eel (Anguilla anguilla) immunity and survival after experimental challenge.

Inland fishery belongs to those branches of animal production that use very large amounts of chemotherapeutics, in particular antibiotics. The accumulation of chemotherapeutic agents in bottom sediments is a direct threat to the aquatic environment and directly affects the condition and health of the fish. Finding a preparation that could be used both prophylactically to increase the resistance of fish and therapeutically in case of infection with pathogenic bacteria, without side effects for fish and aquatic environment could be a great solution to this problem. Our aim was to determine influence of BAFADOR® the new bacteriophage-based preparation on European eel immunity and survival after experimental challenge. Application of BAFADOR® increased total protein level, immunoglobulin level, lysozyme activity and ceruloplasmin level in European eel serum. Potential killing activity and metabolic activity of spleen phagocytes as well as pronephros lymphocyte proliferation of was higher compared to control. The preparation also reduced mortality after experimental infections with the pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. Our results showed that preparation BAFADOR® is well tolerated by the fish organism causing stimulation of cellular and humoral immunity parameters and reduces the mortality of the European eel after experimental challenge.

The composition and structure of the intestinal microflora of Anguilla marmorata at different growth rates: a deep sequencing study.

AIMS: The aim of this study was to determine the intestinal microflora of Anguilla marmorata at different growth rates and to identify potential probiotic/pernicious bacteria. METHODS AND RESULTS: Bacterial communities from eight different eels' intestinal sites (including the intestinal contents and the intestinal mucosa) from three fish groups (three fast-, two medium-, and three stunted-growth samples), two water samples, and one diet sample were characterized by Illumina next-generation sequencing. The data revealed that the predominant genera (relative abundance of bacteria genera >1%) in the intestine of fast- and medium-growth groups were Cetobacterium, Edwardsiella, Clostridium, Lactococcus, Bacteroides, Plesiomonas and Akkermansia. The dominant genus in the stunted-growth group was Spiroplasma. Moreover, culture-associated (water and feed) environmental microbes were distinct from those present in fish intestines, and included Flavobacterium (the dominant bacteria in water) and Corynebacterium (the dominant bacteria in feed). CONCLUSIONS: Only minor differences in gut microbial communities were observed between the fast-growth group and the medium-growth group; however, significant differences were observed between the normal-growth group (including the fast-growth group and medium-growth group, which showed uninhibited growth during the rearing stage) and the stunted-growth group. Together, these data suggested that intestinal microbes were significantly associated with marbled eels' growth rate. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we demonstrated for the first time, the intestinal bacterial communities of A. marmorata at different growth rates. Moreover, we found that the genus Spiroplasma was abundant in the guts of stunted-growth eels, which had never been noticed. Such a finding indicates that the genus Spiroplasma plays a key role associated with retardation in growth and should be controlled to recover the growth of stunted eels, which is meaningful to farmers.

Characterization of MyD88 in Japanese eel, Anguilla japonica.

Myeloid differentiation factor 88 (MyD88) is a key adaptor protein required for the signaling of all Toll-like receptors except TLR3, which results to the interaction of activated TLR complexes via C-terminal TIR domain and the binding of downstream kinase via N-terminal death domain. In this study, the MyD88 gene from the Japanese eel (Anguilla japonica) was identified. The open reading frame of AjMyD88 was 918 bp in length, encoding a protein composed of conserved N-terminal death domain and C-terminal TIR domain, respectively. Multiple alignment revealed highly conserved sites across all examined vertebrate lineages in death and TIR domains. Site-directed mutagenesis and luciferase analysis revealed that the W78A, L91A and L95A mutations in death domain had modest impairment of their ability in activating NF-κB promoter. The expression level of AjMyD88 was investigated by real-time PCR in response to poly I:C stimulation and Edwardsiella tarda infection. Significantly increased MyD88 expression was observed at early phase in all tested tissues/organs in response to E. tarda infection and slight increase was detected in intestine and gill at 16 hpi and in head kidney, spleen and liver at 24 hpi after poly I:C stimulation. Immunofluorescence staining revealed that AjMyD88 is present as condensed forms in the cytoplasm. Taken together, sequence characterization, gene expression and cellular distribution data obtained in this study suggest that AjMyD88, similar to its mammalian ortholog, plays an important role in eel immune response against bacteria.

Community composition, diversity, and metabolism of intestinal microbiota in cultivated European eel (Anguilla anguilla).

The intestinal tract, which harbours tremendous numbers of bacteria, plays a pivotal role in the digestion and absorption of nutrients. Here, high-throughput sequencing technology was used to determine the community composition and complexity of the intestinal microbiota in cultivated European eels during three stages of their lifecycle, after which the metabolic potentials of their intestinal microbial communities were assessed. The results demonstrated that European eel intestinal microbiota were dominated by bacteria in the phyla Proteobacteria and Fusobacteria. Statistical analyses revealed that the three cultured European eel life stages (elver, yellow eel, and silver eel) shared core microbiota dominated by Aeromonas. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) predictions of metagenome function revealed that the European eel intestinal microbiota might play significant roles in host nutrient metabolism. Biolog AN MicroPlate™ analysis and extracellular enzyme assays of culturable intestinal bacteria showed that the intestinal microbiota have a marked advantage in the metabolism of starch, which is the main carbohydrate component in European eel formulated feed. Understanding the ecology and functions of the intestinal microbiota during different developmental stages will help us improve the effects of fish-based bacteria on the composition and metabolic capacity of nutrients in European eels.

Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution.

BACKGROUND: Fish skin mucosal surfaces (SMS) are quite similar in composition and function to some mammalian MS and, in consequence, could constitute an adequate niche for the evolution of mucosal aquatic pathogens in natural environments. We aimed to test this hypothesis by searching for metagenomic and genomic evidences in the SMS-microbiome of a model fish species (Anguilla Anguilla or eel), from different ecosystems (four natural environments of different water salinity and one eel farm) as well as the water microbiome (W-microbiome) surrounding the host. RESULTS: Remarkably, potentially pathogenic Vibrio monopolized wild eel SMS-microbiome from natural ecosystems, Vibrio anguillarum/Vibrio vulnificus and Vibrio cholerae/Vibrio metoecus being the most abundant ones in SMS from estuary and lake, respectively. Functions encoded in the SMS-microbiome differed significantly from those in the W-microbiome and allowed us to predict that successful mucus colonizers should have specific genes for (i) attachment (mainly by forming biofilms), (ii) bacterial competence and communication, and (iii) resistance to mucosal innate immunity, predators (amoeba), and heavy metals/drugs. In addition, we found several mobile genetic elements (mainly integrative conjugative elements) as well as a series of evidences suggesting that bacteria exchange DNA in SMS. Further, we isolated and sequenced a V. metoecus strain from SMS. This isolate shares pathogenicity islands with V. cholerae O1 from intestinal infections that are absent in the rest of sequenced V. metoecus strains, all of them from water and extra-intestinal infections. CONCLUSIONS: We have obtained metagenomic and genomic evidence in favor of the hypothesis on the role of fish mucosal surfaces as a specialized habitat selecting microbes capable of colonizing and persisting on other comparable mucosal surfaces, e.g., the human intestine.

Ribose operon repressor (RbsR) contributes to the adhesion of Aeromonas hydrophila to Anguilla japonica mucus.

The characterization of adhesion between pathogenic bacteria and the host is critical. Pathogenic Aeromonas hydrophila was shown to adhere in vitro to the mucus of Anguilla japonica. To further investigate the adhesion mechanisms of A. hydrophila, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation. Seven mutants that were impaired in adhesion to mucus were selected out of 332 individual colonies, and mutant M196 was the most severely impaired strain. National Center for Biotechnology Information (NCBI) blast analysis showed that mutant M196 was inserted by mini-Tn10 with an ORF of approximately 1,005 bp (GenBank accession numbers KP280172). This ORF is predicted to encode a protein consist of 334 amino acid, which displays the highest identity (98%) with the RbsR of A. hydrophila ATCC 7966. Random inactivation of rbsR gene affected the pleiotropic phenotypes of A. hydrophila. The adhesion ability and the survival level of the rbsR gene mutant (M196) were attenuated compared with the wild-type and rbsR complementary type. The findings of this study indicated that RbsR plays roles in the bacterial adhesion and intracellular survival of A. hydrophila.

Antibiotic resistance and repetitive-element PCR fingerprinting in Aeromonas veronii isolates.

This study evaluated antibiotic resistance and the related genes in total 47 Aeromonas veronii isolates from pet fish, eel (Anguilla japonica) and koi (Cyprinus carpio) in Korea. In comparison with the antibiotic susceptibilities of isolates from eel and koi, those of pet fish were more resistant to ceftiofur, aminoglycosides, tetracycline and nitrofurantoin. And isolates from pet fish showed high prevalences of class 1 integron, quinolones and tetracycline resistance determinants than those from eel and koi. Repetitive-element palindromic PCR (rep-PCR) showed larger diversities among A. veronii isolates. Collectively, pet fish may be a reservoir for multiple clones of A. veronii involved in antibiotic resistance. In this aspect, imported fish in the aquaculture trade should be steadily and continually screened for bacterial antibiotic resistance and related genes.

Incidence of antimicrobial-resistance genes and integrons in antibiotic-resistant bacteria isolated from eels and aquaculture ponds.

The overuse of antimicrobials in aquaculture has promoted the selection of antimicrobial-resistant bacteria. Here we investigated the abundance of antimicrobial-resistance genes and integrons in 108 strains of antibiotic-resistant bacteria isolated from eels and aquaculture ponds in China. Conventional PCR was implemented to examine common antibiotic-resistance genes, integrons, and their gene cassette arrays. The results showed that the antibiotic-resistance genes blaTEM, tetC, sulI, aadA, floR, and qnrB were detected at high percentages, as were a number of other resistance genes. Class I integrons were present in 79.63% of the strains, and 10 out of 108 isolates carried class II integrons. Class III integrons were not detected. Three strains carried both class I and class II integrons, and 73.26% of the class I integron-positive isolates contained the qacEΔ1/sul1 gene. Fourteen types of integron cassette arrays were found among class I integron-positive isolates. A new array, dfrB4-catB3-blaOXA-10-aadA1, was discovered in this study. The gene cassette array dfrA12-orfF-aadA2 was the most widely distributed. In summary, 23 different gene cassettes encoding resistance to 8 classes of antibiotics were identified in the class I integrons, and the main cassettes contained genes encoding resistance to aminoglycosides (aad) and trimethoprim (dfr). All class II integron-positive strains had only a single gene cassette array, viz. dfrA1-catB2-sat2-aadA1. High levels of antimicrobial-resistance genes and integrons in eels and auqauculture ponds suggest that the overuse of antimicrobials should be strictly controlled and that the levels of bacterial antimicrobial-resistance genes in aquaculture should be monitored.

Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

Immunomodulation of Lactobacillus pentosus PL11 against Edwardsiella tarda infection in the head kidney cells of the Japanese eel (Anguilla japonica).

Wild and farm-raised fish can be simultaneously exposed to different types of pathogens in their habitats. Hence, it is important to study their effects, whether isolated or in combination. Therefore, the aim of this study was to evaluate the effects of Lactobacillus pentosus PL11 on the transcription of specific cytokine genes related to immune response, using Japanese eel macrophages as an in vitro model. Head kidney leukocytes were isolated from Japanese eels and cell viability was determined using an MTT reagent. In addition, the Griess reagent was used to determine the nitric oxide (NO) production while, an enzyme-linked immunosobent assay (ELISA) and a quantitative polymerase chain reaction (qPCR) were utilized to quantify the level of proinflammatory cytokines. The results of the study indicated that infection by Edwardsiella tarda alone causes a higher rate of cell death and an increase in the production of proinflammatory cytokines, such as interleukin-1ß (IL-1ß, 822.67 ± 29.48 pg mL(-1)), interleukin-6 (IL-6, 13.57 ± 0.55 pg mL(-1)), and tumor necrosis factor-α (TNF-α, 2033.67 ± 84.68 pg mL(-1)). However, co-culture with L. pentosus PL11 downregulates the production of NO and the related IL-1ß, IL-6, and TNF-α by 46%, 88.4%, 59%, and 77%, respectively. Quantification of the mRNA expression level revealed it to be consistent with the ELISA analysis. Hence, we infer that L. pentosus PL11 plays a significant role in the immunmodulation of the inflammatory responses that arise in fish owing to infection by pathogenic bacteria such as Edwardsiella tarda.

MinD plays an important role in Aeromonas hydrophila adherence to Anguilla japonica mucus.

For a better understanding of the genitic regulation of the adhesion of Aeromonas hydrophila, a mini-Tn10 transposon mutagenesis system was introduced to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOF/Km) and the recipient A. hydrophila strain W. Out of 332 individual colonies, 7 mutants with significantly attenuated adhesion ability were selected. The DNA sequence flanking the mini-Tn10 transposon inserted in the mutant strain M45 was amplified by TAIL-PCR. The results showed that an ORF of approximately 870 bp (GenBank accession number KP271930) of the mutant strain M45 was inserted by mini-Tn10. This ORF putatively encodes a deduced 289 amino acid protein that displays the highest identity (100%) with the CobQ/CobB/MinD/ParA family protein of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type W, the mutant strain M45 and the complemented strain C45 were investigated. The results indicated that mutation of MinD gene in A. hydrophila could lead to abnormal cell division and reduce the ability of adhesion, biofilm formation and bacterial motility.