Search

Aquaculture has suffered significant financial losses as a result of the infection of zoonotic Aeromonas hydrophila, which has a high level of resistance to classic antibiotics. In this study, we isolated an A. hydrophila strain B3 from diseased soft-shelled turtle (Pelodiscus sinensis), which is one of the most commercially significant freshwater farmed reptiles in East Asia, and found that A. hydrophila was its dominant pathogen. To better understand the inhibition effect and action mechanism of Chinese herbs on A. hydrophila, we conducted Chinese herbs screening and found that Lonicera japonica had a significant antibacterial effect on A. hydrophila B3. Experimental therapeutics of L. japonica on soft-shelled turtle showed that the supplement of 1% L. japonica to diet could significantly upregulate the immunity-related gene expression of soft-shelled turtle and protect soft-shelled turtle against A. hydrophila infection. Histopathological section results validated the protective effect of L. japonica. As the major effective component of L. japonica, chlorogenic acid demonstrated significant inhibitory effect on the growth of A. hydrophila with MIC at 6.4 mg/mL. The in vitro assay suggested that chlorogenic acid could inhibit the hemolysin/protease production and biofilm formation of A. hydrophila and significantly decrease the expression of quorum sensing, biofilm formation, and hemolysin-related genes in A. hydrophila. Our results showed that the Chinese herb L. japonica would be a promising candidate for the treatment of A. hydrophila infections in aquaculture, and it not only improves the immune response of aquatic animals but also inhibits the virulence factor (such as biofilm formation) expression of A. hydrophila. KEY POINTS: • A. hydrophila was the dominant pathogen of the diseased soft-shelled turtle. • L. japonica can protect soft-shelled turtle against A. hydrophila infection. • Chlorogenic acid inhibits the growth and biofilm formation of A. hydrophila., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Edwardsiella anguillarum and Aeromonas hydrophila are two common bacterial pathogens affecting cultivated eels, and the differences in their virulence remain unclear. In this study, after two groups of American eels (Anguilla rostrata) were administered the LD 50 dose of E. anguillarum and A. hydrophila, respectively, the histopathology of the liver, trunk kidney, and spleen, as well as transcriptomic RNA sequencing (RNA-seq) analysis of the spleen, was examined at three time points: pre-infection (Con group) and post-infection at 36 h (Ea_36 group, Ah_36 group) and 60 h (Ea_60 group, Ah_60 group). The results showed that the differences in pathological changes were characterized by severe hepatocyte edema at 36 h post-infection (hpi) and hepatocyte atrophy at 60 hpi in the livers of eels infected by A. hydrophila, in contrast to the severe atrophy of glomeruli in the trunk kidneys and numerous bacterial nodules in the spleens of eels infected by E. anguillarum. The RNA-seq results revealed 906 and 77 typical differentially expressed genes (DEGs) in eels infected with E. anguillarum and A. hydrophila, respectively, compared to the control eels. The DEGs between the infected and control groups were predominantly annotated in GO terms related to binding, catalytic activity, membrane part, cell part, and cellular process, as well as in KEGG pathways associated with human diseases and organismal systems. The GO enrichment analysis showed 83 and 146 differential GO terms, along with 32 and 78 differential KEGG pathways in two comparisons of Ea_36 vs Con versus Ah_36 vs Con and Ea_60 vs Con versus Ah_60 vs Con, respectively. Furthermore, the analysis of differential alternative splicing genes (DASs) showed 1244 and 1341 DASs out of 12,907 and 12,833 AS genes, respectively, in the comparisons of Ea_36 vs Ah_36 and Ea_60 vs Ah_60. These DASs were enriched in two common KEGG pathways: "NOD-like receptor signaling pathway" and "necroptosis" which shared 11 hub DASs. Finally, analysis of protein-protein interactions revealed that 91 of 412 cross DASs between Ea_36 vs Ah_36 and Ea_60 vs Ah_60 potentially play an essential role in the difference in virulence of E. anguillarum and A. hydrophila in American eels, with 12 encoded proteins being particularly notable. Together, this study is the first to report a comparative pathogenicity and RNA-seq analysis of E. anguillarum and A. hydrophila in American eels, shedding new light on our understanding of the differences in virulence as revealed by pathological changes, DEGs, and DASs, contributing to more effective control strategies to prevent outbreaks of bacterial infections., Competing Interests: Declarations. Ethics Approval: The fish used in this study were cultivated under protocols approved by the Jimei University Animal Care Committee (2011–59), and all experiments complied with the current laws of China and international organizations for the use of experimental animals. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The present study was conducted to study the composition of gut microbiome in the advanced fingerling and fingerling stage of striped pangasius catfish and catla during healthy and diseased conditions. Healthy pangasius and catla fishes were obtained from commercial farms and injected with the LD 50 dose of A. hydrophila. The intestinal samples from the control and injected group were collected and pooled for 16 s metagenomic analysis. Community analysis was performed by targeting the 16 s rRNA gene to explore and compare the gut microbiota composition of these fishes. The operational taxonomic units (OTUs) consisted of four major phyla: Bacteroidia, Proteobacteria, Firmicutes, and Actinobacteria. Alpha and beta diversity indices were carried out to understand the diversity of microbes within and between a sample. While comparing the advanced fingerling and fingerling stage gut microbiome of Pangasius catfish, the dominance of Proteobacteria was found in fingerlings, whereas Firmicutes and Bacteroides were found in advanced fingerlings. In catla, Proteobacteria and Bacteroides were predominant. Taxonomic abundance of the microbiota in control and diseased Pangasius and catla fishes at phylum, class, order, family, genus, and species levels were also depicted. The present study is the first of its kind, and it will help to identify the diversity of novel potential bacterial species involved in disease protection of fishes. It can lead to the development of sustainable prophylactic measures against (re-)emerging bacterial diseases in aquaculture., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Aeromonas hydrophila, a prevalent pathogen in the aquaculture industry, poses significant challenges due to its drug-resistant strains. Moreover, residues of antibiotics like streptomycin, extensively employed in aquaculture settings, drive selective bacterial evolution, leading to the progressive development of resistance to this agent. However, the underlying mechanism of its intrinsic adaptation to antibiotics remains elusive. Here, we employed a quantitative proteomics approach to investigate the differences in protein expression between A. hydrophila under streptomycin (SM) stress and nonstress conditions. Notably, bioinformatics analysis unveiled the potential involvement of metal pathways, including metal cluster binding, iron-sulfur cluster binding, and transition metal ion binding, in influencing A. hydrophila's resistance to SM. Furthermore, we evaluated the sensitivity of eight gene deletion strains related to streptomycin and observed the potential roles of petA and AHA_4705 in SM resistance. Collectively, our findings enhance the understanding of A. hydrophila's response behavior to streptomycin stress and shed light on its intrinsic adaptation mechanism., (© 2024 Wiley‐VCH GmbH.)

Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch's postulates, challenge experiments were conducted and median lethal dose (LD 50 ) of L. garvieae and A. hydrophila was calculated as 1 × 10 5.91 CFU per mL and 1 × 10 5.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

This study used berberine hydrochloride to treat the Asian paddle crab, Charybdis japonica infected with the Gram-negative bacterium Aeromonas hydrophila at concentrations of 0, 100, 200 and 300 mg/L. The effect of berberine hydrochloride on the survival rate and gut microbiota of C. japonica was investigated. Berberine hydrochloride improved the stability of the intestinal flora, with an increase in the abundance of probiotic species and a decrease in the abundance of both pathogenic bacteria after treatment with high concentrations of berberine hydrochloride. Berberine hydrochloride altered peroxidase activity (POD), malondialdehyde (MDA), and lipid peroxidation (LPO) in the intestinal tract compared to the control. Berberine hydrochloride could modulate the energy released from the enzyme activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the intestinal tract of C. japonica infected with A. hydrophila. Zona occludens 1 (ZO-1), Zinc finger E-box binding homeobox 1 (ZEB1), occludin and signal transducer, and activator of transcription5b (STAT5b) expression were also increased, which improved intestinal barrier function. The results of this study provide new insights into the role of berberine hydrochloride in intestinal immune mechanisms and oxidative stress in crustaceans., (© 2024. The Author(s).)

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila , a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170  A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tekedar, Patel, Blom, Griffin, Waldbieser, Kumru, Abdelhamed, Dharan, Hanson and Lawrence.)

Aeromonas hydrophila is not a traditional intracellular bacterium. However, previous studies revealed that pathogenic A. hydrophila B11 could temporarily survive for at least 24 h in fish phagocytes, and the regulation of intracellular survival in bacteria was associated with regulators of the LuxR-type. The mechanisms of luxR 08110 on the A. hydrophila's survival in macrophages were investigated using comprehensive transcriptome analysis and biological phenotype analysis in this study. The results showed that after luxR 08110 was silenced, the intracellular survival ability of bacteria was significantly diminished. Comparative transcriptome analysis revealed that luxR 08110 was a critical regulator of A. hydrophila, which regulated the expression of over 1200 genes, involving in bacterial flagellar assembly and chemotaxis, ribosome, sulphur metabolism, glycerolipid metabolism, and other mechanisms. Further studies confirmed that after the inhibition of expression of luxR 08110 , the motility, chemotaxis and adhesion of A. hydrophila significantly decreased. Moreover, compared with the wild-type strain, the survival rates of silencing strain were all considerably reduced under both H 2 O 2 and low pH stress conditions. According to both transcriptome analysis and phenotypic tests, the luxR 08110 of A. hydrophila could act as global regulator in bacteria intracellular survival. This regulator regulated intracellular survival of A. hydrophila mainly through two ways. One way is to regulate bacterial flagellar synthesis and further affects the motility, chemotaxis and adhesion of bacteria. The other way is to regulate sulphur and glycerolipid metabolisms, thus affecting bacterial energy production and the ability to resist environmental stress., (© 2024 John Wiley & Sons Ltd.)

Aeromonas hydrophila, an opportunistic warm water pathogen, has always been a threat to aquaculture, leading to substantial economic losses. Vaccination of the cultured fish would effectively prevent Aeromoniasis, and recent advancements in nanotechnology show promise for efficacious vaccines. Oral delivery would be the most practical and convenient method of vaccine delivery in a grow-out pond. This study studied the immunogenicity and protective efficacy of a nanoparticle-loaded outer membrane protein A from A. hydrophila in the zebrafish model. The protein was over-expressed, purified, and encapsulated using poly lactic-co-glycolic acid (PLGA) nanoparticles via the double emulsion method. The PLGA nanoparticles loaded with recombinant OmpA (rOmpA) exhibited a size of 295 ± 15.1 nm, an encapsulation efficiency of 72.52%, and a polydispersity index of 0.292 ± 0.07. Scanning electron microscopy confirmed the spherical and isolated nature of the PLGA-rOmpA nanoparticles. The protective efficacy in A. hydrophila-infected zebrafish after oral administration of the nanovaccine resulted in relative percentage survival of 77.7. Gene expression studies showed significant upregulation of immune genes in the vaccinated fish. The results demonstrate the usefulness of oral administration of nanovaccine-loaded rOmpA as a potential vaccine since it induced a robust immune response and conferred adequate protection against A. hydrophila in zebrafish, Danio rerio., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Although the genus Aeromonas inhabits the natural environment, it has also been isolated from hospital patient specimens as a causative agent of Aeromonas infections. However, it is not known whether clinical strains live in the natural environment, and if these strains have acquired antimicrobial resistance. In this study, we performed the typing of flagellin A gene (flaA) of clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using Polymerase Chain Reaction (PCR) assay with newly designed primers. Detection rates of the clinical and environmental flaA types of A. hydrophila were 66.7% and 88.2%, and the corresponding rates for A. veronii biovar sobria were 66.7% and 90.9%. The PCR assays could significantly discriminate between clinical and environmental strains of both species in approximately 4 h. Also, among the 63 clinical Aeromonas strains used, only one extended-spectrum β-lactamase-producing bacteria, no plasmid-mediated quinolone resistance bacteria, and only four multidrug-resistant bacteria were detected. Therefore, the PCR assays could be useful for the rapid diagnosis of these Aeromonas infections and the monitoring of clinical strain invasion into water-related facilities and environments. Also, the frequency of drug-resistant Aeromonas in clinical isolates from Okinawa Prefecture, Japan, appeared to be low., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

LysR-type transcriptional regulators (LTTRs) are ubiquitously distributed and abundant transcriptional regulators in prokaryotes, playing pivotal roles in diverse physiological processes. Nonetheless, despite their prevalence, the intricate functionalities and physiological implications of this protein family remain incompletely elucidated. In this study, we employed a comprehensive approach to deepen our understanding of LTTRs by generating a collection of 20 LTTR gene-deletion strains in Aeromonas hydrophila, accounting for 42.6 % of the predicted total LTTR repertoire, and subjected them to meticulous assessment of their physiological phenotypes. Leveraging quantitative proteomics, we conducted a comparative analysis of protein expression variations between six representative mutants and the wild-type strain. Subsequent bioinformatics analysis unveiled the involvement of these LTTRs in modulating a wide array of biological processes, notably including two-component regulatory systems (TCSs) and intracellular central metabolism. Moreover, employing subsequent microbiological methodologies, we experimentally verified the direct involvement of at least six LTTRs in the regulation of galactose metabolism. Importantly, through ELISA and competitive ELISA assays, we demonstrated the competitive binding capabilities of these LTTRs with the promoter of the α-galactosidase gene AHA_1897 and identified that four LTTRs (XapR, YidZ, YeeY, and AHA_1805) do not engage in competitive binding with other LTTRs. Overall, our comprehensive findings not only provide fundamental insights into the regulatory mechanisms governing crucial physiological functions of bacteria through LTTR family proteins but also uncover an intricate and interactive regulatory network mediated by LTTRs., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Herein, we detected one multidrug-resistant Aeromonas hydrophila strain K522 co-carrying two bla KPC -2 genes together with a novel chromosomal integrative and mobilizable element (IME) Tn7548 from China. To reveal the genetic characteristics of the novel reservoir of bla KPC-2 and IME in Aeromonas, a detailed genomic characterization of K522 was performed, and a phylogenetic analysis of Tn7412-related IMEs was carried out., Methods: Carbapenemases were detected by using the immunocolloidal gold technique and antimicrobial susceptibility was tested by using VITEK 2. The whole-genome sequences of K522 were analysed using phylogenetics, detailed dissection, and comparison., Results: Strain K522 carried a Tn7412-related chromosomal IME Tn7548 and three resistance plasmids pK522-A-KPC, pK522-B-KPC, and pK522-MOX. A phylogenetic tree of 82 Tn7412-related IMEs was constructed, and five families of IMEs were divided. These IMEs shared four key backbone genes: int, repC, and hipAB, and carried various profiles of antimicrobial resistance genes (ARGs). pK522-A-KPC and pK522-B-KPC carried bla KPC-2 and belonged to IncG and unclassified type plasmid, respectively. The bla KPC-2 regions of these two plasmids were the truncated version derived from Tn6296, resulting in the carbapenem resistance of K522., Conclusion: We first reported A. hydrophila harbouring a novel Tn7412-related IME Tn7548 together with two bla KPC-2 carrying plasmids and a MDR plasmid. Three of these four mobile genetic elements (MGEs) discovered in A. hydrophila K522 were novel. The emergence of novel MGEs carrying ARGs indicated the rapid evolution of the resistance gene vectors in A. hydrophila under selection pressure and would contribute to the further dissemination of various ARGs in Aeromonas., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aeromonas hydrophila is an opportunistic motile pathogen with a broad host range, infecting both terrestrial and aquatic animals. Environmental and geographical conditions exert selective pressure on both geno- and phenotypes of pathogens. Flagellin, directly exposed to external environments and containing important immunogenic epitopes, may display significant variability in response to external conditions. In this study, we conducted a comparative analysis of ~  150 A. hydrophila genomes, leading to the identification of six subunits of the flagellin gene (fla-1 to fla-4, flaA, and flaB). Individual strains harbored different composition of flagellin subunits and copies. The composition of subunits showed distinct patterns depending on environmental sources. Strains from aquatic environments were mainly comprised of fla-1 to fla-4 subunits, while terrestrial strains predominated in groups harboring flaA and flaB subunits. Each flagellin showed varying levels of expression, with flaA and flaB demonstrating significantly higher expression compared to others. One of the chemotaxis pathways that control flagellin movement through a two-component system was significantly upregulated in flaA(+ 1)/flaB(+ 1) group, whereas flaA and flaB showed different transcriptomic expressions. The genes positively correlated with flaA expression were relevant to biofilm formation and bacterial chemotaxis, but flaB showed a negative correlation with the genes in ABC transporters and quorum sensing pathway. However, the expression patterns of fla-2 to fla-4 were identical. This suggests various types of flagellin subunits may have different biological functions. The composition and expression levels of flagellin subunits could provide valuable insights into the adaptation of A. hydrophila and the differences among strains in response to various external environments., (© 2024. The Author(s).)

Aeromonas hydrophila , an aquatic pathogenic bacterium, has been found to infect many fish species and cause huge aquaculture losses. Antibiotics are the most common drugs used to treat these infections. However, antibiotic abuse can lead to the development of antibiotic resistance. Probiotics have the potential to replace antibiotics for preventing infections. Zebrafish ( Danio rerio ) is a model organism used to study the innate immune system and host-pathogen interactions. Currently, there is little information on how the fish immune system responds to A. hydrophila and probiotic treatment. To increase the understanding of the molecular mechanisms behind the zebrafish defense against A. hydrophila and provide evidence that antibiotics can be replaced by probiotics, a transcriptome analysis of the zebrafish spleen was conducted 48 hours after infection by A. hydrophila , as well as after treatment using Lactococcus lactis KUST48 4 hours after infection. A total of 36,499 genes were obtained. There were 3,337 genes found to have significant differential expression between treatment and control groups. According to further annotation and enrichment analysis, differentially expressed genes (DEGs) were involved in signal transduction, endocrine system cancer, and the immune system. Insulin resistance disappeared in the zebrafish after treatment. Quantitative real-time PCR was performed to confirm the significant regulation of immune defense DEGs, the results of which were consistent with the RNA-sequencing data. These results could serve as a basis for future studies on the immune response to A. hydrophila and provide suggestions for probiotic alternatives to antibiotics, which will be of great significance to aquaculture and environmental protection.IMPORTANCEIn recent years, the unreasonable use of antibiotics has led to the emergence of drug-resistant pathogenic bacteria, antibiotic residues, cross infection, toxic side effects, and so on, which has caused a serious threat to human food safety and life health. In recent years, many studies have demonstrated the potential of probiotics as a substitute for antibiotics, but there is still a lack of understanding of the molecular mechanisms underlying probiotic therapy. We conduct a research on the impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila -infected zebrafish spleen. Mortality of zebrafish infected with A. hydrophila was significantly reduced after treatment with L. lactis KUST48. Our results can help to strengthen our understanding of the pathogenic mechanisms of zebrafish and provide a valuable reference for the molecular mechanisms of probiotic therapy., Competing Interests: The authors declare no conflict of interest.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sripradite et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antimicrobial peptides (AMPs), which are widely present in animals and plants, have a broad distribution, strong broad-spectrum antibacterial activity, low likelihood of developing drug resistance, high thermal stability and antiviral properties. The present study investigated the effects of adding AMPs from Hermetia illucens larvae on the growth performance, muscle composition, antioxidant capacity, immune response, gene expression, antibacterial ability and intestinal microbiota of Cherax quadricarinatus (red claw crayfish). Five experimental diets were prepared by adding 50 (M1), 100 (M2), 150 (M3) and 200 (M4) mg/kg of crude AMP extract from H. illucens larvae to the basal diet feed, which was also used as the control (M0). After an eight-week feeding experiment, it was discovered that the addition of 100-150 mg/kg of H. illucens larvae AMPs to the feed significantly improved the weight gain rate and specific growth rate of C. quadricarinatus. Furthermore, the addition of H. illucens larvae AMPs to the feed had no significant effect on the moisture content, crude protein, crude fat and ash content of the C. quadricarinatus muscle. The addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed also increased the antioxidant capacity, nonspecific immune enzyme activity and related gene expression levels in C. quadricarinatus, thereby enhancing their antioxidant capacity and immune function. The H. illucens larvae AMPs improved the structure and composition of the intestinal microbiota of C. quadricarinatus, increasing the microbial community diversity of the crayfish gut. Finally, the addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed enhanced the resistance of C. quadricarinatus against Aeromonas hydrophila, improving the survival rate of the crayfish. Based on the aforementioned findings, it is recommended that H. illucens larvae AMPs be incorporated into the C. quadricarinatus feed at a concentration of 100-150 mg/kg., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila., Competing Interests: The authors have declared that no competing interests, (Copyright: © 2024 Mahmood et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the present study, Aeromonas hydrophila was isolated from Tilapia zillii and Mugil cephalus samples collected during different seasons from various Suez Canal areas in Egypt. The prevalence of A. hydrophila, virulence genes, and antibiotic resistance profile of the isolates to the commonly used antibiotics in aquaculture were investigated to identify multiple drug resistance (MDR) and extensive drug-resistant (XDR) strains. In addition, a pathogenicity test was conducted using A. hydrophila, which was isolated and selected based on the prevalence of virulence and resistance genes, and morbidity of natural infected fish. The results revealed that A. hydrophila was isolated from 38 of the 120 collected fish samples (31.6%) and confirmed phenotypically and biochemically. Several virulence genes were detected in retrieved A. hydrophila isolates, including aerolysin aerA (57.9%), ser (28.9%), alt (26.3%), ast (13.1%), act (7.9%), hlyA (7.9%), and nuc (18.4%). Detection of antibiotic-resistant genes revealed that all isolates were positive for bla pse1 (100%), bla SHV (42.1%), tetA (60.5%), and sul1 (42.1%). 63.1% of recovered isolates were considered MDR, while 28.9% of recovered isolates were considered XDR. Some isolates harbor both virulence and MDR genes; the highest percentage carried 11, followed by isolates harboring 9 virulence and resistance genes. It could be concluded that the high prevalence of A. hydrophila in aquaculture species and their diverse antibiotic resistance and virulence genes suggest the high risk of Aeromonas infection and could have important implications for aquaculture and public health., (© 2024. The Author(s).)

Background: Aeromonas hydrophila infections can cause gastrointestinal symptoms such as diarrhea; however, deep infections are rarely reported. Outbreaks of A. hydrophila are reported more frequently in fish, poultry, and snakes than in humans. This study aimed to track clonal relatedness of deep infections caused by A. hydrophila using whole genome sequencing (WGS)., Methods: We collected three isolates of A. hydrophila in July 19 to August 29, 2019, from patients that underwent spine surgery. Accurate species identification was performed using whole-genome average nucleotide identity (ANI). Antimicrobial susceptibility testing was performed using a VITEK 2 automated AST-N334 Gram-negative susceptibility card system. Antimicrobial resistance and virulence genes were identified using the Comprehensive Antibiotic Resistance Database and Virulence Factor Database VFanalyzer., Results: All three isolates were identified as A. hydrophila based on ANI and multilocus sequence typing analysis revealed that A. hydrophila belonged to a novel sequence type (ST1172). All three isolates were susceptible to amikacin and levofloxacin; however, they were resistant to piperacillin/tazobactam, ceftriaxone, cefuroxime, cefoxitin, and imipenem. Isolate 19W05620 (patient 3) showed increased ceftazidime resistance (minimum inhibitory concentration ≥ 64 µg/mL). All three isolates possessed the same chromosomally encoded β-lactamases, including bla OXA-724 (β-lactamase), imiH (metallo-β-lactamase), and bla MOX-13 (AmpC) in plasmids., Conclusions: Our study validated the transmission of a novel carbapenem-resistant A. hydrophila sequence type (ST1172) in patients that underwent spine surgery. Control measures should be developed to prevent dissemination of A. hydrophila in the hospital setting., (© 2024. The Author(s).)

Sichuan is a significant aquaculture province in China, with a total aquaculture output of 1.72 × 10 6 tons in 2022. One of the most significant microorganisms hurting the Sichuan aquaculture is Aeromonas hydrophila, whose genotype and antibiotic resistance are yet unknown. This study isolated a total of 64 strains of A. hydrophila from various regions during September 2019 to June 2021 within Sichuan province, China. The technique of Multi-Locus Sequence Typing (MLST) was used for the purpose of molecular typing. Meanwhile, identification of antibiotic resistance phenotype and antibiotic resistance gene was performed. The findings of the study revealed that 64 isolates exhibited 29 sequence types (ST) throughout different regions in Sichuan, with 25 of these ST types being newly identified. Notably, the ST251 emerged as the predominant sequence type responsible for the pandemic. The resistance rate of isolated strains to roxithromycin was as high as 98.3%, followed by co-trimoxazole (87.5%), sulfafurazole (87.5%), imipenem (80%), amoxicillin (60%), and clindamycin (57.8%). Fifteen strains of A. hydrophila exhibited resistance to medicines across a minimum of three categories, suggesting the development of multidrug resistance in these isolates. A total of 63 ARGs were detected from the isolates, which mediated a range of antibiotic resistance mechanisms, with deactivation and efflux potentially serving as the primary mechanisms of antibiotic resistance. This study revealed the diversity of A. hydrophila genotypes and the risk of antibiotic resistance in Sichuan, providing reference for scientific and effective control of A. hydrophila infection., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Background: Aquatic opportunistic pathogen Aeromonas hydrophila, known to persist in low-nutrient chlorinated waters, can cause life-threatening infections. Two intensive care units experienced a cluster of Aeromonas infections following outdoor temperature spikes coinciding with recurrent plumbing issues, with fatalities due to severe underlying comorbidities co-occurring with extensively-drug resistant (XDR) Aeromonas., Methods: We investigated this cluster using whole genome sequencing to assess genetic relatedness of isolates and identify antimicrobial resistance determinants. Three A. hydrophila were isolated from patients staying in or adjacent to rooms with plumbing issues during or immediately after periods of elevated outdoor temperatures. Sinks and faucets were swabbed for culture., Results: All A. hydrophila clinical isolates exhibited carbapenem resistance but were not genetically related. Diverse resistance determinants corresponding to extensively-drug resistant were found, including co-occurring KPC-3 and VIM-2, OXA-232, and chromosomal CphA-like carbapenemase genes, contributing to major treatment challenges. All 3 patients were treated with multiple antibiotic regimens to overcome various carbapenemase classes and expired due to underlying comorbidities. Environmental culture yielded no Aeromonas., Conclusions: While the investigation revealed no singular source of contamination, it supports a possible link between plumbing issues, elevated outdoor temperatures and incidence of nosocomial Aeromonas infections. The diversity of carbapenemase genes detected in these wastewater-derived Aeromonas warrants heightened infection prevention precautions during periods of plumbing problems especially with heat waves., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aeromonas hydrophila is a serious human and animal co-pathogenic bacterium. Flagellum, a key virulence factor, is vital for bacterium tissue colonization and invasion. flgL is a crucial gene involved in the composition of flagellum. However, the impact of flgL on virulence is not yet clear. In this study, we constructed a stable mutant strain (△flgL-AH) using homologous recombination. The results of the attack experiments indicated a significant decrease in the virulence of △flgL-AH. The biological properties analysis revealed a significant decline in swimming ability and biofilm formation capacity in △flgL-AH and the transmission electron microscope results showed that the ∆flgL-AH strain did not have a flagellar structure. Moreover, a significant decrease in the adhesion capacity of ∆flgL-AH was found using absolute fluorescence quantitative polymerase chain reaction (PCR). The quantitative real-time PCR results showed that the expression of omp and the eight flagellum-related genes were down-regulated. In summary, flgL mutation leads to a reduction in pathogenicity possibly via decreasing the swimming ability, biofilm formation capacity and adhesion capacity, these changes might result from the down expression of omp and flagellar-related genes., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Aeromonas hydrophila and methicillin-resistant Staphylococcus aureus (MRSA) are potent bacterial pathogens posing major hazards to human health via consuming fish harboring these pathogens or by cross-contamination beyond the contaminated environment. The aim of this study was to determine risk variables associated with the presence of certain pathogenic bacteria from Mugil cephalus fish in retail markets in Egypt. The virulence genes of A. hydrophila and S. aureus were also studied. Furthermore, the antibiotic sensitivity and multidrug resistance of the microorganisms were evaluated., Methods: In a cross-sectional investigation, 370 samples were collected from mullet skin and muscle samples, washing water, fish handlers, knives, and chopping boards. Furthermore, fish handlers' public health implications were assessed via their response to a descriptive questionnaire., Results: S. aureus and Aeromonas species dominated the investigated samples with percentages of 26.76% and 30.81%, respectively. Furthermore, A. hydrophila and MRSA were the predominant recovered bacterial pathogens among washing water and knives (53.85% and 46.66%, respectively). The virulence markers aerA and hlyA were found in 90.7% and 46.5% of A. hydrophila isolates, respectively. Moreover, the virulence genes nuc and mec were prevalent in 80% and 60% of S. aureus isolates, respectively. Antimicrobial susceptibility results revealed that all A. hydrophila isolates were resistant to amoxicillin and all MRSA isolates were resistant to amoxicillin and ampicillin. Remarkably, multiple drug resistance (MDR) patterns were detected in high proportions in A. hydrophila (88.37%) and MRSA (100%) isolates. The prevalence of Aeromonas spp. and S. aureus had a positive significant correlation with the frequency of handwashing and use of sanitizer in cleaning of instruments. MRSA showed the highest significant prevalence rate in the oldest age category., Conclusion: The pathogenic bacteria recovered in this study were virulent and had a significant correlation with risk factors associated with improper fish handling. Furthermore, a high frequency of MDR was detected in these pathogenic bacteria, posing a significant risk to food safety and public health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Attia, Abou Elez, El-Gazzar, Elnahriry, Alfifi, Al-Harthi, Alkhalifah, Hozzein, Diab and Ibrahim.)

Mandarin fish (Siniperca chuatsi) is a valuable freshwater fish species widely cultured in China. Its aquaculture production is challenged by bacterial septicaemia, which is one of the most common bacterial diseases. Antimicrobial peptides (AMPs) play a critical role in the innate immune system of fish, exhibiting defensive and inhibitory effects against a wide range of pathogens. This study aimed to identify the antimicrobial peptide genes in mandarin fish using transcriptomes data obtained from 17 tissue in our laboratory. Through nucleotide sequence alignment and protein structural domain analysis, 15 antimicrobial peptide genes (moronecidin, pleurocidin, lysozyme g, thymosin β 12 , hepcidin, leap 2, β-defensin, galectin 8, galectin 9, apoB, apoD, apoE, apoF, apoM, and nk-lysin) were identified, of which 9 antimicrobial peptide genes were identified for the first time. In addition, 15 AMPs were subjected to sequence characterization and protein structure analysis. After injection with Aeromonas hydrophila, the number of red blood cells, hemoglobin concentration, and platelet counts in mandarin fish showed a decreasing trend, indicating partial hemolysis. The expression change patterns of 15 AMP genes in the intestine after A. hydrophila infection were examined by using qRT-PCR. The results revealed, marked up-regulation (approximately 116.04) of the hepcidin gene, down-regulation of the piscidin family genes expression. Moreover, most AMP genes were responded in the early stages after A. hydrophila challenge. This study provides fundamental information for investigating the role of the different antimicrobial peptide genes in mandarin fish in defense against A. hydrophila infection., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Aeromonas hydrophila , a Gram-negative zoonotic bacterium, causes high mortality in fish farming and immunocompromised patients. This study aimed to extract methyl gallate (MG) from the flowers of Camellia nitidissima Chi and evaluate its potential as a quorum sensing inhibitor (QSI) against Aeromonas hydrophila SHAe 115. MG reduced QS-associated virulence factors, including hemolysis, protease, and lipase, while impairing swimming motility and biofilm formation. Additionally, MG down-regulated positive regulatory genes ( ahyR , fleQ ) and up-regulated negative regulators ( litR , fleN ). This highlights MG's promise as a potent QSI for A. hydrophila SHAe 115, advancing strategies against infections in aquaculture and human health.

Naturally infected Channa punctata exhibiting bacterial septicemic syndrome including ulcerations along with mortality records were collected from a fish farm in Assam during winter season (early November 2020 to early January 2021). The moribund fishes were subjected for bacterial isolation followed by identification of the bacteria. Two dominant emerging bacterial pathogens were identified as Aeromonas veronii (isolate ZooGURD-01) and Aeromonas hydrophila (isolate ZooGURD-05) by standard biochemical characterization and 16S rRNA and rpo B gene amplification. Re-infection experiments of both the bacterial isolates in healthy disease-free C. punctata showed similar symptoms to that of natural infection thus confirming their virulence. The LD 50 calculated during challenge test for both the isolates ZooGURD-01 and ZooGURD-05 found to be pathogenic at 2.6 × 10 4 and 1.6 × 10 4  CFU/fish respectively. Further PCR amplification of specific virulent genes (aerolysin, hemolysin and enterotoxin) confirmed pathogenicity for both isolates. Histopathological examinations of liver and kidney in re-infection experiments showed prominent changes supporting bacterial septicaemia. Antibiotic sensitivity pattern showed that the isolates ZooGURD-01 and ZooGURD-05 were sensitive to 22 and 19 out of 25 antimicrobials respectively. The present study was the first report on the mortality of farmed C. punctata associated with natural infection caused by A. veronii and A. hydrophila with no record of pathogenicity of A. veronii in C. punctata., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Aeromonas hydrophila, a Gram-negative pathogenic bacterium, is responsible for huge economic losses in aquaculture. In this study, we evaluated the efficacy of bacteriophage AHPMCC7 which was isolated by using A. hydrophila MTCC 1739 as a host. This bacteriophage exhibited 10 min latent period and burst size was 275. In liquid culture, bacteriophage AHPMCC7 could completely lyse A. hydrophila MTCC 1739 after 2 h. AHPMCC7 genome was 42,277 bp long with 58.9% G + C content. The genome consisted of 48 CDSs and no tRNA. The comparative genomic analyses clearly implied that AHPMCC7 might represent a novel species of the genus Aphunavirus under Autographiviridae family. Bacteriophage AHPMCC7 could survive at broad pH (3-10), temperature (4-37 °C), and salinity (0-40 ppt). In aquarium trial, AHPMCC7 could control A. hydrophila MTCC 1739 without affecting the survivability of Litopenaeus vannamei. Clearly, the bacteriophage AHPMCC7 might be used in shrimp aquaculture as a biocontrol agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Aeromonas hydrophila is an important pathogen that mainly harms aquatic animals and exhibits resistance to a variety of antibiotics. This study investigated the effect of epigallocatechin-3-gallate (EGCG) on the virulence factors of A.hydrophila and its impact on adhesion, invasion, and cytotoxicity in Caco-2 cells. The potential mechanism of antibacterial activity of EGCG was investigated by transcriptomic analysis., Results: EGCG not only inhibited the production of biofilm, hemolytic activity, motility, and protease activity of A.hydrophila , but also reduced its adhesion, invasion, and cytotoxicity in Caco-2 cells. Transcriptomic analysis indicated that the antimicrobial activity of EGCG may be achieved by weakening the chemotaxis and stress response of the bacteria, as well as inhibiting the TonB system. Animal studies demonstrated that EGCG can significantly improve the survival rate and organs damage of zebrafish infected with A.hydrophila ., Conclusion: EGCG would be a potential alternative drug for the prevention and treatment of A. hydrophila infections by anti-virulence mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yin, Yan, Cheng, Zhang, Li, Hu, Gao, Chen, Tang and Luo.)

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein that triggers downstream cascades mediated by both TNFR and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF6 is involved in various biological processes, including innate and adaptive immunity. In the present study, a homolog of TRAF6 from Macrobrachium rosenbergii (MrTRAF6) was identified and characterized. The full-length cDNA of MrTRAF6 consisted of 2,114 nucleotides with an open reading frame (ORF) of 1,695 nucleotides encoding a 564-amino acid protein that contained a conserved TRAF family motif including two RING-type zinc fingers and a C-terminal meprin and TRAF homology (MATH) domain. The putative amino sequence of MrTRAF6 shared 45.5-97.3% identity with TRAF6s from other crustacean species with the highest identity to Macrobrachium nipponense TRAF6. Phylogenetic analysis revealed that MrTRAF6 was closely related to TRAF6 of invertebrates and clustered with crustaceans. According to gene expression analysis, the MrTRAF6 transcript demonstrated broad expression in all tissues tested, with the highest expression level in gill and the lowest in muscle tissues. Upon immune challenge with Aeromonas hydrophila, significant upregulation of MrTRAF6 expression was found in the gill, hepatopancreas, hemocyte, and muscle. Furthermore, an RNA interference assay showed that silencing MrTRAF6 by dsRNA could reduce the expression of mannose-binding lectin (MBL) and crustin, but no significant change was detected in anti-lipopolysaccharide factor 5 (ALF5) levels. In addition, the cumulative mortality rate of MrTRAF6-silenced M. rosenbergii was significantly increased after A. hydrophila infection. These findings indicated that MrTRAF6 is involved in antibacterial activity and plays a critical role in the innate immune response of M. rosenbergii., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1 H and 13 C NMR spectroscopy, including 1 H, 1 H NOESY, and 1 H, 13 C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined: [Formula: see text] The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes. For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Aeromonas hydrophila is a zoonotic bacterial pathogen that frequently causes disease and mass mortalities among cultured and feral fishes worldwide. In Ethiopia, A. hydrophila outbreak was reported in Sebeta fish ponds and in Lake Tana fishery. However, there is no to little information on the molecular, and phenotypical characteristics of A. hydrophila in Ethiopian fisheries. Therefore, a cross-sectional study was conducted from November 2020 to May 2021 in selected Ethiopian Rift valley lakes., Results: A total of 140 samples were collected aseptically from fish (Muscle, Gill, Intestine, Spleen and Kidney) from fish landing sites, market and restaurants with purposive sampling methods. Aeromonas selective media (AMB), morphological and biochemical tests were used to isolate and identify A. hydrophila. Accordingly, the pathogen was isolated from 81 (60.45%) of samples. Among the isolates 92.59% expressed virulence trait through β hemolysis on blood agar media with 5% sheep blood. Moreover, 54 strains (66.67%) were further confirmed with Real-Time PCR (qPCR) using ahaI gene specific primers and optimized protocol. The highest (68.51%) were detected from live fish, (24.07%) were from market fish and the lowest (7.4%%) were from ready-to-eat products. Antibiogram analysis was conducted on ten representative isolates. Accordingly, A. hydrophila isolates were susceptible to ciprofloxacin (100%), chloramphenicol (100%) and ceftriaxone (100%). However, all ten isolates were resistant to Amoxicillin and Penicillin., Conclusions: The study indicates A. hydrophila strains carrying virulence ahaI gene that were ß-hemolytic and resistant to antibiotics commonly used in human and veterinary medicine are circulating in the fishery. The detection of the pathogen in 140 of the sampled fish population is alarming for potential outbreaks and zoonosis. Therefore, further molecular epidemiology of the disease should be studied to establish potential inter host transmission and antibiotic resistance traits. Therefore, raising the public awareness on risk associated with consuming undercooked or raw fish meat is pertinent., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Rapid and accurate detection of bacterial pathogens is critical in controlling disease outbreaks affecting farmed fish. The present study aimed to develop a novel serological diagnostic approach using nano‑silver based Enzyme-linked immunosorbent assay (ELISA) for speedy detection of Aeromonas veronii infections in Nile tilapia. A. veronii isolates used in ELISA assays were recovered from moribund Nile tilapia during a disease outbreak in a private fish farm in Egypt. A. veronii isolates were identified based on alignment analysis of the gyrB and 16S rRNA gene sequences. A. veronii antisera used in ELISA assays were prepared in tilapia, and the bacterial antigens were formalin-killed. The cut-off values were 0.46 and 0.48 in traditional and nano-based ELISA. There were no cross-reactions with bacterial isolates (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas fluorescens, and Vibrio vulnificus). The lowest antigen concentration that produced positive results after checkerboard titration in indirect-ELISA (i-ELISA) and dot ELISA was 15 μg and 250 ng of prepared antigen, respectively. Nano-ELISA and nano-based dot-ELISA antigen concentration was 10 μg and 100 ng, respectively. Sera concentration was 1:100 in indirect-ELISA and dot-ELISA, while it was 1:50 in nano-based ELISA and nano dot-ELISA. The i-ELISA successfully detected anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity, while in the dot-ELISA, the sensitivity and specificity were 83.33% and 100%, respectively. Nano dot-ELISA had 100% sensitivity, specificity, and accuracy. Nano dot-ELISA assays have higher specificity, sensitivity, and accuracy than traditional ELISAs in detecting A. veronii. Further studies are needed to develop a rapid test kit for on-site field diagnosis., Competing Interests: Declaration of Competing Interest All authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The proliferation of antibiotic-resistant bacteria and antimicrobial resistance is a pressing public health challenge because of their possible transfer to humans via contact with polluted water sources. In this study, three freshwater resources were assessed for important physicochemical characteristics as well as heterotrophic and coliform bacteria and as potential reservoirs for extended-spectrum beta-lactamase (ESBL) strains. The physicochemical characteristics ranged from 7.0 to 8.3; 25 to 30 °C, 0.4 to 93 mg/L, 0.53 to 8.80 mg/L and 53 to 240 mg/L for pH, temperature, dissolved oxygen (DO), biological oxygen demand (BOD 5 ) and total dissolved solids, respectively. The physicochemical characteristics mostly align with guidelines except for the DO and BOD 5 in some instances. Seventy-six (76) Aeromonas hydrophila and 65 Escherichia coli O157: H7 isolates were identified by preliminary biochemical analysis and PCR from the three sites. Among these, A. hydrophila displayed higher frequencies of antimicrobial resistance, with all 76 (100%) isolates completely resistant to cefuroxime and cefotaxime and with MARI ≥ 0.61. The test isolates showed more than 80% resistance against five of the ten test antimicrobials, with resistance against cefixime, a cephalosporin antibiotic being the highest at 95% (134/141). The frequency of the detection of the resistance genes in the A. hydrophila isolates generally ranged between 0% (bla SHV ) and 26.3% (bla CTX-M ), while the frequency of detection among the E. coli O157:H7 isolates ranged between 4.6% (bla CTX-M ) and 58.4% (bla TEM ). Our findings indicate that the distribution of antibiotic-resistant bacteria with diverse ESBL-producing capabilities and virulence genes in freshwater sources potentially threatens public health and the environment., (© 2023. The Author(s).)

Aquaculture plays an increasingly important if not critical role in the current and future world food supply. Aeromonas hydrophila, a heterotrophic, Gram-negative, bacterium found in fresh or brackish water in warm climates poses a serious threat to the aquaculture industry in many areas, causing significant economic losses. Rapid, portable detection methods of A. hydrophila are needed for its effective control and mitigation. We have developed a surface plasmon resonance (SPR) technique to detect PCR (polymerase chain reaction) products that can replace agarose gel electrophoresis, or otherwise provide an alternative to costlier and more complicated real-time, fluorescence-based detection. The SPR method provides sensitivity comparable to gel electrophoresis, while reducing labor, cross-contamination, and test time, and employs simpler instrumentation with lower cost than real-time PCR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The colicin I receptor (CirA) is a well-studied outer membrane protein that has been reported to play important roles in antibiotic resistance, virulence, and iron homeostasis, although its exact physiological roles require further investigation. In this study, differentially expressed proteins between the ΔahcirA and wild-type (WT) strains of Aeromonas hydrophila were compared using quantitative proteomics. Bioinformatics analysis revealed that the expression of peptide, histidine, and arginine ATP-binding cassette (ABC) transporter system-related proteins was significantly higher in the ΔahcirA strain. Subsequent growth assays revealed that ΔahcirA grew slower than the WT strain in nutrient-limited medium when supplemented with dipeptide, histidine, and arginine as the carbon source. Far-western blot analysis further confirmed that AhCirA can directly bind to histidine/arginine and dipeptide small-molecule substrates in addition to their periplasmic-binding proteins, AhDppA and AhHisJ, respectively. These results indicate that AhCirA may play an important role in the uptake of amino acids and peptides as a channel-forming porin while also directly interacting with ABC transporters to transport nutrient substances into the plasma membrane. Overall, this study demonstrates that AhCirA is a multifunctional protein in A. hydrophila and extends our understanding of known nutrient transport mechanisms among bacteria., (© 2023 Applied Microbiology International and John Wiley & Sons Ltd.)

Recently, the prevalence of Aeromonas hydrophila antibiotic-resistant strains has been reported in aquaculture, but its intrinsic antibiotic resistance mechanisms are largely unknown. In the present study, a label-free proteomics technology was used to compare the differential protein abundances in response to norfloxacin (NOR) stress in A. hydrophila . The results showed that there were 186 proteins decreasing and 220 proteins increasing abundances in response to NOR stress. Bioinformatics analysis showed that the differentially expressed proteins were enriched in several biological processes, such as sulfur metabolism and homologous recombination. Furthermore, the antibiotic sensitivity assays showed that the deletion of AHA&#95;0904 , cirA , and cysI significantly decreased the resistance against NOR, whereas Δ AHA&#95;1239 , ΔcysA , Δ cysD , and Δ cysN significantly increased the resistance against NOR. Our results provide insights into NOR resistance mechanisms and indicate that AHA&#95;0904 , cirA , AHA&#95;1239 , and sulfur metabolism may play important roles in NOR resistance in A. hydrophila .

Background: Aeromonas hydrophila is an opportunistic pathogen. Thus, it has received significant attention mainly in the fish sectors with high production scales. Nile tilapia broodstock confined in the environment of fish hatcheries can be stressed. Hence, they are vulnerable to A. hydrophila., Results: Sequencing of the gyr B gene revealed the presence of 18 different A. hydrophila strains (kdy 10,620-10,637), which were deposited in the NCBI under accession numbers ON745861-ON745878. The median lethal doses of the isolates ranged from 2.62 × 10 4 to 3.02 × 10 6  CFU/mL. Antibiotic resistant genes, sulfonamide (sul1) and tetracycline (tetA) were found in the eighteen isolates. Approximately 83.3% of A. hydrophila strains were sensitive to ciprofloxacin and florfenicol. Further, eight A. hydrophila strains had high MDR indices at 0.27-0.45. All isolates presented with hemolysin activity. However, only 72.22% of them had proteolytic activity, and only 61.11% could form biofilms. Bacterial isolates harbored different pattern virulence genes, the heat-stable cytotonic enterotoxin (ast), cytotoxic enterotoxin (act), and hemolysin (hly) genes were the most prevalent. Also, a trial to inhibit bacterial growth was conducted using titanium dioxide nanoparticles (TiO 2 NPs) with three sizes (13, 32, and 123 nm). If A. hydrophila strains with a high MDR index were tested against TiO 2 NPs (20 µg/mL) for 1, 12, and 24 h, those with a small size had a greater bactericidal action than large ones. Bacterial strains were inhibited at different percentages in response to TiO 2 NP treatment., Conclusions: Nile tilapia broodstock, mortality is associated with different A. hydrophila strains, which harbored virulent and MDR genes. Furthermore, TiO 2 NPs had bactericidal activity, thereby resulting in a considerable reduction in bacterial load., (© 2023. The Author(s).)

Background: Aeromonas hydrophila is an important foodborne and zoonotic pathogen causing serious diseases. Hence, revealing the pathogenic mechanism of A. hydrophila will be of importance in the development of novel therapies. Aeromonas hydrophila litR was reported to be regulated by two quorum sensing (QS) pathways, indicating that it is involved in QS network regulation correlated with bacterial virulence. However, the function of LitR is currently not understood. Therefore, we aimed to reveal the potential regulatory mechanisms of LitR on virulence-related genes., Methods and Results: In this study, amino acid sequences analysis of LitR was conducted, providing bioinformatics evidence for its function as a potential transcriptional regulator. LitR protein was heterologous expressed, purified and its in-vitro multimeric forms were observed with gel filtration chromatography. The correlation between intracellular LitR expression level and cell density was analyzed with immunoblots. Regulation mechanisms of LitR on several important virulence-related factors were investigated with qRT-PCR, EMSA, DNase I footprinting and microscale thermophoresis binding assays, etc. Results showed that recombinant LitR protein aggregated mainly as dimer and hexamer in vitro. Intracellular expression level of LitR was positively correlated with cell density of A. hydrophila. Furthermore, LitR exhibited complicated regulation modes on virulence-related genes; it could directly bind to promoter regions of the hemolysin, serine protease and T6SS effector protein VgrG encoded genes. The promoter region of the hemolysin gene showed high binding affinity and mainly two binding sites for LitR. Different dissociation constants were obtained for LitR interaction with the hemolysin gene binding motifs I and II. Assays focusing on physiological characteristics of A. hydrophila prove that LitR positively regulated hemolytic and total extracellular protease activities., Conclusions: This study investigated the function of LitR as a quorum sensing transcriptional regulator in regulation of virulence-related genes, which will help reveal the mechanisms of A. hydrophila pathogenicity. LitR could serve as a potential target for development of new antimicrobial agents from the perspective of QS regulation., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Tilapia is a primary aquaculture fish in Thailand, but little is known about the occurrence of antimicrobial resistance (AMR) in Aeromonas hydrophila , Salmonella spp., and Vibrio cholerae colonizing healthy tilapia intended for human consumption and the co-occurrence of these AMR bacteria in the cultivation water., Methods: This study determined the phenotype and genotype of AMR, extended-spectrum β -lactamase (ESBL) production, and virulence factors of A. hydrophila , Salmonella spp., and V. cholerae isolated from hybrid red tilapia and cultivation water in Thailand. Standard culture methods such as USFDA's BAM or ISO procedures were used for the original isolation, with all isolates confirmed by biochemical tests, serotyping, and species-specific gene detection based on PCR., Results: A total of 278 isolates consisting of 15 A. hydrophila , 188 Salmonella spp., and 75 V. cholerae isolates were retrieved from a previous study. All isolates of A. hydrophila and Salmonella isolates were resistance to at least one antimicrobial, with 26.7% and 72.3% of the isolates being multidrug resistant (MDR), respectively. All A. hydrophila isolates were resistant to ampicillin (100%), followed by oxytetracycline (26.7%), tetracycline (26.7%), trimethoprim (26.7%), and oxolinic acid (20.0%). The predominant resistance genes in A. hydrophila were mcr-3 (20.0%), followed by 13.3% of isolates having floR , qnrS , sul1 , sul2 , and dfrA1. Salmonella isolates also exhibited a high prevalence of resistance to ampicillin (79.3%), oxolinic acid (75.5%), oxytetracycline (71.8%), chloramphenicol (62.8%), and florfenicol (55.3%). The most common resistance genes in these Salmonella isolates were qnrS (65.4%), tetA (64.9%), bla TEM (63.8%), and floR (55.9%). All V. cholerae isolates were susceptible to all antimicrobials tested, while the most common resistance gene was sul1 (12.0%). One isolate of A. hydrophila was positive for int1 , while all isolates of Salmonella and V. cholerae isolates were negative for integrons and int SXT . None of the bacterial isolates in this study were producing ESBL. The occurrence of mcr-3 (20.0%) in these isolates from tilapia aquaculture may signify a serious occupational and consumer health risk given that colistin is a last resort antimicrobial for treatment of Gram-negative bacteria infections., Conclusions: Findings from this study on AMR bacteria in hybrid red tilapia suggest that aquaculture as practiced in Thailand can select for ubiquitous AMR pathogens, mobile genetic elements, and an emerging reservoir of mcr and colistin-resistant bacteria. Resistant and pathogenic bacteria, such as resistance to ampicillin and tetracycline, or MDR Salmonella circulating in aquaculture, together highlight the public health concerns and foodborne risks of zoonotic pathogens in humans from cultured freshwater fish., Competing Interests: The authors declare there are no competing interests., (©2023 Thaotumpitak et al.)

Objectives: Aeromonas spp. often cause life-threatening diseases, including necrotizing fasciitis, which may lead to septic shock and ultimately death. Aeromonas infections are believed to be transmitted via minor wounds or the consumption of fresh fish. However, after the detection of Aeromonas hydrophila in ticks in areas endemic to Japanese-spotted fever (JSF), a novel transmission route of A. hydrophila (i.e., via tick bites) has been proposed. We investigated the prevalence of A. hydrophila in ticks in areas endemic and not endemic to JSF in the Mie Prefecture, Japan., Methods: We collected ticks from endemic and nonendemic areas in summer and winter and assessed them for presence of A. hydrophila using polymerase chain reaction., Results: Six A. hydrophila isolates were obtained from 95 ticks in endemic areas, whereas one A. hydrophila isolate was obtained from 142 ticks in non-endemic areas, in summer. All ticks that harboured A. hydrophila were Haemaphysalis longicornis (H.L); these ticks were almost at the larval stage and also carried Rickettsia spp. in the endemic area. In contrast, 51 and 41 ticks in the endemic and non-endemic areas were captured in winter, respectively; A. hydrophila was not detected in these., Conclusions: This study revealed the prevalence of tick-borne A. hydrophila. Therefore, the risk of transmission of A. hydrophila via a tick bite should be considered in the following conditions: areas abundant in H. L. harbouring Rickettsia spp., in areas endemic for JSF, presence of ticks in the larval stage and during the summer season., (© 2022 John Wiley & Sons Ltd.)

The collectin subfamily member 11 (Colec11), plays an important role in innate immunity as a pattern recognition molecule. In the present study, a colec11 homolog was identified and characterised from Qihe crucian carp, namely, Ca-colec11. The full-length cDNA of Ca-colec11 was composed of 1129 bp, with a 99 bp 5'-untranslated region (UTR), 816 bp open reading frame (ORF) encoding a 271-aa protein and 214 bp 3'-UTR with a polyadenylation signal sequence (aataaa) and a poly(A) tail. The deduced amino acid sequence of Ca-Colec11 contained a si gnal peptide, collagen domain, neck region and carbohydrate-recognition domain (CRD), which had four conserved cysteine residues (Cys 170 -Cys 256 and Cys 242 -Cys 264 ) and an EPN/WND motif required for carbohydrate-binding specificity. Tissue expression profile analysis by quantitative real-time polymerase chain reaction (RT-qPCR) showed that Ca-colec11 was ubiquitously distributed in the tested tissues and highly expressed in the liver. The gene expression levels of Ca-colec11 were evidently up-regulated in the liver, spleen, kidney and head kidney after infection with A. hydrophila and S. aureus. The recombinant Ca-Colec11 (rCa-Colec11) purified from Escherichia coli BL21 (DE3) could agglutinate A. hydrophila and S. aureus, and it possessed haemagglutination activity against rabbit erythrocytes, which was inhibited by various carbohydrates, including d-Mannose, N-Acetyl-d-mannosamine, l-Fucose, d-Glucose, N-Acetyl-d-glucosamine, d-Galactose, LPS and PGN. Furthermore, rCa-Colec11 could inhibit the growth of A. hydrophila and S. aureus. These findings collectively demonstrated that Ca-Colec11, as a PRR, could play a role in the immune defence of Qihe crucian carp., Competing Interests: Declaration of competing interest All authors have no competing interest to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mannan oligosaccharides (MOS) are functional oligosaccharides with beneficial effects on the non-specific immunity of Megalobrama amblycephala , but systematic studies on the immunomodulatory mechanisms of MOS are still lacking. To investigate the protective mechanisms of three different levels of dietary MOS supplementation on the intestinal immunity of juvenile M. amblycephala , comparative digital gene expression (DGE) profiling was performed. In this study, 622 differentially expressed genes (DEGs) were identified, while the similar expression tendency of 34 genes by qRT-PCR validated the accuracy of the DGE analyses. Gene Ontology (GO) enrichment revealed that the DEGs were mainly enriched in two functional categories of biological process and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were mainly related to complement and coagulation cascades, coagulation cascades, platelet activation, natural killer cell mediated cytotoxicity, Fc gamma R-mediated phagocytosis and antigen processing and presentation. In addition, the pro-inflammatory, apoptosis and tight junction-related genes were more significantly up-regulated upon infection in the dietary MOS groups to enhance host immune functions and maintain the stability of the intestinal barrier. These results will be helpful to clarify the regulatory mechanism of MOS on the intestinal immunity of M. amblycephala and lay the theoretical foundation for the prevention and protection of fish bacterial diseases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: Aeromonas hydrophila is an opportunistic gram-negative bacillus that causes diseases such as gastroenteritis, muscle infections, soft tissue, sepsis, and skin diseases in humans. Today, the prevalence of antibiotic resistance in bacteria has led to treatment failure and prolonged treatment. Therefore, the aim of this study was to evaluate the level of antibiotic resistance in isolates carrying bla VIM, bla KPC, bla NDM, bla IMP, bla SHV, bla TEM, bla CTX-M and class I and II integrons in Aeromonas hydrophila., Methods: In this cross-sectional study, Aeromonas hydrophila were collected from different clinical specimens in Hazrat Masoumeh Hospital, Qom Province, Iran, from 2018 to 2020. The collected isolates were identified by standard biochemical tests. Then, using specific primers bla VIM, bla KPC, bla NDM, bla IMP, bla SHV, bla TEM, bla CTX-M genes, and class I and II integrons were evaluated by PCR method. Then, data were analyzed using SPSS software and chi-squared tests, and the significance level was determined as p ≤ 0.05., Results: During the sample collection period, 100 Aeromonas hydrophila were collected. Based on the results of the antibiotic resistance pattern, the highest and lowest rate of antibiotic resistance to ampicillin (92%) and azithromycin (4%) were determined for both. Among the 100 isolates, 60 isolates produced broad-spectrum beta-lactamase (ESBL) and 50 isolates produced carbapenemase. Among the studied beta-lactamase genes, the highest and lowest frequencies were related to bla CTX-M (58%) and bla TEM (1%), respectively. The frequency of class I and II integron genes was 27% and 15%, respectively., Conclusions: The results of the study of antibiotic resistance, beta-lactamase, and carbapenemase genes showed high resistance in Aeromonas hydrophila, which raises concerns with regard to controlling infection in medical centers. Also, the study of antibiotic resistance in the presence of beta-lactamase genes showed that there was only a significant relationship between the presence of bla CTX-M gene and resistance to imipenem (p = 0.037).

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.)

Private wells are used daily worldwide as convenient household water sources. In Japan, where water supply coverage is high, well water is occasionally used for non-potable purposes, such as irrigation and watering. Currently, the main microbiological test of well water is designed to detect Escherichia coli, which is an indicator of fecal contamination, using culture methods. Water use such as watering generates bioaerosols, which may cause airborne infection. However, many causative bacteria of aerosol-derived infections, such as Legionella spp., are difficult to detect using culture methods. Thus, more comprehensive modern assessment is desirable for securing the microbiological quality of well water. Here, the bacterial community structure of five private wells located in different environments was examined using the rapid and portable MinION sequencer, which enabled us to identify bacteria to the species level based on full-length 16S ribosomal RNA (rRNA) gene sequences. The results revealed the differences in the bacterial community structures of water samples from the five wells and detected Legionella pneumophila and Aeromonas hydrophila as new candidate microbial indicators. The comprehensive analysis method used in this study successfully detected bacteria causing opportunistic infections, which are difficult to detect by conventional methods. This approach is expected to be routinely applied in the future as a highly accurate method for assessing the microbiological quality of private well water.

Protozoan predation has been demonstrated to be a strong driving force for bacterial defence strategies in the environment. Our previous study demonstrated that Aeromonas hydrophila NJ-35, which evolved small-colony variants (SCVs), displayed various adaptive traits in response to Tetrahymena thermophila predation, such as enhanced phage resistance. However, the evolutionary mechanisms are largely unknown. In this study, we performed a genome- and transcriptome-wide analysis of the SCV1, representing one strain of the SCVs, for identification of the genes of mutation and altered expression underlying this phage resistance phenotype. Our study demonstrated that phage resistance caused by T. thermophila predation was due to the downregulation of a flagellar biosynthesis regulator, flhF , in SCV1. Interestingly, we confirmed that phage resistance in SCV1 was not straightforwardly attributable to the absence of flagella but to FlhF-mediated secretion of extracellular protein that hinders phage adsorption. This finding improves our understanding of the mechanisms by which A. hydrophila lowers the susceptibility to phage infection under predation pressure, and highlights an important contribution of bacterium-protozoan interactions in driving the adaptive evolution of pathogens in complex environments.

The molecular mechanisms underlying Aeromonas hydrophila -pathogenesis are not well understood. Using head kidney macrophages (HKM) of Clarias gariepinus , we previously reported the role of ER-stress in A. hydrophila -induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca 2+ imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in A. hydrophila- infected HKM. CHOP promotes HKM apoptosis by inhibiting AKT activation and enhancing JNK signaling. Elevated mitochondrial ROS (mtROS) was recorded which declined significantly by ameliorating ER-stress and in the presence of ER-Ca 2+ release modulators (2-APB and dantrolene) and mitochondrial-Ca 2+ uptake inhibitor, Ru360, together suggesting the role of ER-mitochondrial Ca 2+ dynamics in mtROS generation. Inhibiting mtROS production reduced HKM death implicating the pro-apoptotic role of mtROS in A. hydrophila -pathogenesis. The expression of autophagic proteins (LC3B, beclin-1, and atg 5) was suppressed in the infected HKM. Our results with autophagy-inducer rapamycin demonstrated that impaired autophagy favored the cytosolic accumulation of mitochondrial DNA (mtDNA) and the process depended on mtROS levels. Enhanced caspase-1 activity and IL-1β production was detected and transfection studies coupled with pharmacological inhibitors implicated mtROS/mtDNA axis to be crucial for activating the caspase-1/IL-1β cascade in infected HKM. RNAi studies further suggested the involvement of IL-1β in generating pro-apoptotic NO in A. hydrophila -infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating A. hydrophila pathogenesis. Based on our observations, we conclude that A. hydrophila induces ER-stress and inhibits mitophagy resulting in mitochondrial dysfunction which leads to mtROS production and translocation of mtDNA into cytosol triggering the activation of caspase-1/IL-1β-mediated NO production, culminating in HKM apoptosis.

The immunogenicity of Aha1, an OMP of Aeromonas hydrophila mediating the adhesion of bacteria onto the mucosal surface of hosts has been established. In this study, recombinant vectors, pPG1 and pPG2, carrying a 1366 bp DNA fragment that was responsible for encoding the 49 kDa Aha1 from A. hydrophila were constructed, respectively, then electroporated into a probiotic strain Lactobacillus casei CC16 separately to generate two types of recombinants, L. casei- pPG1-Aha1 (Lc-pPG1-Aha1) and L. casei -pPG2-Aha1 (Lc-pPG2-Aha1). Subsequently, these were orally administered into common carps to examine their immunogenicity. The expression and localization of the expressed Aha1 protein relative to the carrier L. casei was validated via Western blotting, flow cytometry, and immune fluorescence separately. The recombinant vaccines produced were shown high efficacies, stimulated higher level of antibodies and AKP, ACP, SOD, LZM, C3, C4 in serum in hosts. Immune-related gene expressions of cytokines including IL-10, IL-1β, TNF-α, IFN-γ in the livers, spleens, HK, and intestines were up-regulated significantly. Besides, a more potent phagocytosis response was observed in immunized fish, and higher survival rates were presented in common carps immunized with Lc-pPG1-Aha1 (60%) and Lc-pPG2-Aha1 (50%) after re-infection with virulent strain A. hydrophila . Moreover, the recombinant L. casei were shown a stronger propensity for survivability in the intestine in immunized fish. Taken together, the recombinant L. casei strains might be promising candidates for oral vaccination against A. hydrophila infections in common carps.

In this paper, the whole genome of the multidrug-resistant Aeromonas hydrophila MX16A was comprehensively analyzed and compared after sequencing by PacBio RS II. To shed light on the drug resistance mechanism of A. hydrophila MX16A, a Kirby-Bauer disk diffusion method was used to assess the phenotypic drug susceptibility. Importantly, resistance against β-lactam, sulfonamides, rifamycins, macrolides, tetracyclines and chloramphenicols was largely consistent with the prediction analysis results of drug resistance genes in the CARD database. The varied types of resistance genes identified from A. hydrophila MX16A revealed multiple resistance mechanisms, including enzyme inactivation, gene mutation and active effusion. The publicly available complete genomes of 35 Aeromonas hydrophila strains on NCBI, including MX16A, were downloaded for genomic comparison and analysis. The analysis of 33 genomes with ANI greater than 95% showed that the pan-genome consisted of 9556 genes, and the core genes converged to 3485 genes. In summary, the obtained results showed that A. hydrophila exhibited a great genomic diversity as well as diverse metabolic function and it is believed that frequent exchanges between strains lead to the horizontal transfer of drug resistance genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guo, Zeng, Ma, Cai, Jiang, Zhai, Xu and Lin.)

Aeromonas hydrophila infections are common in aquaculture. Our previous studies found that the A. hydrophila B11 strain can survive in fish macrophages for at least 24 h and the two-component system EnvZ/OmpR may be involved in intracellular survival. To reveal the role and mechanism of the two-component system EnvZ/OmpR in intracellular survival of A. hydrophila, the genes of envZ/ompR were silenced by shRNAi. The results showed that the survival rates of the envZ-RNAi and ompR-RNAi strains were only 2.05% and 3.75%, respectively, which were decreased by 91% and 83.6% compared with that of the wild-type strain. The escape ability of envZ-RNAi and ompR-RNAi was also decreased by 51.4% and 19.7%, respectively. The comparative transcriptome analysis revealed that the functional genes directly related to bacterial intracellular survival mainly included the genes related to anti-stress capacity, and the genes related to Zn 2+ and Mg 2+ transport. Further research confirmed that two-component system EnvZ/OmpR can regulate the expression of the important molecular chaperones, such as groEL, htpG, dnaK, clpB and grpE. The expression of these molecular chaperones in wild-type strain was up-regulated with the increase in H 2 O 2 concentrations, while the expression of these molecular chaperones in silent strains did not change significantly. Cells that phagocytosed wild-type strain had higher ROS content than cells that phagocytosed silent strains. Two-component system EnvZ/OmpR could also regulate zinc transporter (znuA, znuB, znuC) and zinc efflux protein (zntA) to maintain zinc homeostasis in cells, thus affecting the ability of bacteria to survive in phagocytes. Moreover, two-component system EnvZ/OmpR could affect the growth and intracellular survival of A. hydrophila by regulating the expression of MgtA, MgtC and MgtE and participating in bacterial Mg 2+ homeostasis in fish macrophages., (© 2022 John Wiley & Sons Ltd.)