Motile Aeromonas Septicemia Research Articles

Deteksi Gen Resistensi Aeromonas hydrophila yang Diisolasi dari Budidaya Ikan Lele Di Jambi

Aeromonas hydrophila is one of the causative agents for Motile Aeromonas Septicemia (MAS) in catfish. MAS treatment is generally carried out by administering antibiotics. This study aimed to determine the antibiotic resistance profile of A. hydrophila and detect the presence of resistance genes. The samples are archived A. hydrophila isolates isolated from catfish ponds in the district of Muara Jambi, Bungo, Jambi Province, and Merangin total of 18 isolates. Testing the antibiotic resistance profile was carried out using the disk diffusion method against tetracycline, oxytetracycline, ampicillin, amoxicillin, ciprofloxacin, enrofloxacin, and streptomycin. The detected resistance genes were ampC, qnrA, qnrB, and qnrS. The results showed A. hydrophila isolates were resistant to tetracycline (100%), oxytetracycline (94.42%), ampicillin (77.70%), and amoxicillin (77.72%) and still sensitive to ciprofloxacin (83.32%), enrofloxacin (100 %), and streptomycin (100%). The ampC gene was found in 8 out of 14 isolates resistant to beta-lactam antibiotics (57.14%). The qnrA and qnrS genes were found in 3 out of 4 isolates resistant to quinolone antibiotics (75%), while qnrB was found in 1 isolate (25%). A. hydrophila isolated from catfish ponds in Jambi develops resistance to antibiotics and resistance genes found in isolates resistant to antibiotics.

Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan

Abstract Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.

RESPON IKAN NILA (OREOCHROMIS NILOTICUS) DAN IKAN LELE (CLARIAS GARIEPINUS) YANG DIINFEKSI AEROMONAS HYDROPHILLA DENGAN EMPAT TINGKAT PASASE

Tilapia and catfish are some of the freshwater fisheries commodities that occupy the top rank in terms of the amount of production produced. Disease attacks in Tilapia and Catfish cultivation such as Motile Aeromonas Septicemia (MAS) can be detrimental to fish farmers. Motile Aeromonas Septicemia (MAS) disease is caused by the bacteria Aeromonas hydrophilla. This research aims to determine the differences in the physical condition of Tilapia and Catfish which were injected with Aeromonas hydrophilla bacteria and had been treated using the Koch Postulate method four times to reach 50% fish mortality. In this study, the Tilapia and Catfish samples used were 6-8 cm in size with a bacterial infection dose of 0.1ml/fish. Tilapia showed clinical symptoms of thin fins and the injection site began to become inflamed, while catfish experienced bleeding and wounds. Catfish tend to experience more serious injuries after exposure to A. hydrophilla compared to Tilapia due to differences in response to disease symptoms. Tilapia fish show symptoms of loose scales due to bacterial infections such as Aeromonas dhakensis, Pseudomonas mosselii, and Microbacterium paraoxydans.

Identification and characterization of motile Aeromonas spp. isolated from farmed Nile tilapia (Oreochromis niloticus) in the Philippines.

Motile Aeromonas septicaemia (MAS) caused by motile Aeromonas species is an important disease in farmed freshwater fish due to intensification of culture and improper farm practices. This study characterized and profiled motile Aeromonas species recovered from clinically sick tilapia farmed in the Philippines, with a view to identifying targeted disease prevention and control measures against MAS in farmed tilapia species. Sixteen isolates from diseased farmed Nile tilapia were identified as Aeromonas veronii (n=14), Aeromonas caviae (n=1), and Aeromonas dhakensis (n=1). Five biochemical profiles using API 20E were exhibited by the A. veronii strains giving an unreliable identification. A high level of agreement was observed in identifying the Aeromonas strains using 16S rRNA and rpoD gene sequencing, although the latter has a higher discriminatory value. Three or more virulence genes dominated by cytotoxic enterotoxin act and aerolysin aer were detected. Different genotypes based on virulence gene clustering suggested varied mechanisms used by Aeromonas to colonize and infect or to mutualistically co-exist with the fish. Acquired multiple antibiotic resistance was found in a single A. veronii isolate. All were susceptible to enrofloxacin, oxolinic acid, florfenicol, and chloramphenicol. Tetracycline and sulfonamide resistances and class 1 integron were detected in three A. veronii isolates. Several strains of motile aeromonads, especially A. veronii, which have varied genotypes based on virulence, biochemical profile, and antibiotic resistance, are involved in MAS in natural disease outbreaks in farmed Nile tilapia in the Philippines.

The potential of shallot skin powder and actinomycetes metabolites as antimicrobe and antibiofilm in the treatment of eel (Anguilla bicolor bicolor) infected with Aeromonas hydrophila

BackgroundEel (Anguilla bicolor bicolor) is an Indonesian export commodity. However, it is facing a problem related to Aeromonas hydrophila, which can cause motile aeromonas septicemia (MAS) and produce biofilm formation. Problem with antibiotic resistance challenges the need of an alternative treatment. Therefore, it is important to explore a solution to treat infection and the biofilm formed by A. hydrophila.ObjectivesIn this study, we used shallot skin powder and actinomycetes metabolite 20 PM as antimicrobe and antibiofilm to treated eels infected with A. hydrophila.ResultsShallot skin powder (6.25 g 100 g−1 feed) and Actinomycetes 20 PM metabolite (2 mL 100 g−1 feed) were found to be effective as antimicrobe and antibiofilm agent in treating eels infected with A. hydrophila. Eel treated with antibiotic, shallot skin powder, and actinomycetes metabolite had 80%, 66%, and 73% survival rates, respectively. Other indicators such as red blood cell count, hemoglobin, and hematocrit were increased, but white blood cell count and phagocytic activity were dropped. Biofilm destruction were analyzed using scanning electron microscopy to determined antibiofilm activity of actinomycetes metabolite against biofilm of A. Hydrophila.ConclusionsShallot skin powder and actinomycetes metabolite were potential to treat infection of A. hydrophila in eel as an alternative treatment to antibiotics.

Comparative genomic analysis of Aeromonas dhakensis and Aeromonas hydrophila from diseased striped catfish fingerlings cultured in Vietnam.

Motile Aeromonas septicemia (MAS) is a burden for striped catfish (Pangasius hypophthalmus) farmers in Vietnam. MAS can be caused by several species of Aeromonas but Aeromonas hydrophila is seen as the leading cause of MAS in aquaculture, but recent reports suggest that A. dhakensis is also causing MAS. Here we investigated the bacterial etiology of MAS and compared the genomic features of A. hydrophila and A. dhakensis. We collected 86 isolates from diseased striped catfish fingerlings over 5 years from eight provinces in Vietnam. Species identification was done using PCR, MALDI-TOF and whole genome sequence (WGS). The MICs of commonly used antimicrobials was established. Thirty presumed A. hydrophila isolates were sequenced for species confirmation and genomic comparison. A phylogenetic analysis was conducted using publicly available sequences and sequences from this study. A total of 25/30 isolates were A. dhakensis sequence type (ST) 656 and 5/30 isolates were A. hydrophila ST 251. Our isolates and all publicly available A. hydrophila isolates from Vietnam belonged to ST 251 and differed with <200 single nucleotide polymorphisms (SNP). Similarly, all A. dhakensis isolates from Vietnam belonged to ST 656 and differed with <100 SNPs. The tet(A) gene was found in 1/5 A. hydrophila and 19/25 A. dhakensis. All A. hydrophila had an MIC ≤2 mg/L while 19/25 A. dhakensis had MIC ≥8 mg/L for oxytetracycline. The floR gene was only found in A. dhakensis (14/25) which showed a MIC ≥8 mg/L for florfenicol. Key virulence genes, i.e., aerA/act, ahh1 and hlyA were present in all genomes, while ast was only present in A. dhakensis. This study confirms previous findings where A. dhakensis was the dominating pathogen causing MAS and that the importance of A. hydrophila has likely been overestimated. The differences in antimicrobial susceptibility between the two species could indicate a need for targeted antimicrobial treatment plans. The lipopolysaccharide regions and outer membrane proteins did not significantly differ in their immunogenic potentials, but it remains to be determined with in vivo experiments whether there is a difference in the efficacy of available vaccines against A. hydrophila and A. dhakensis.

Isolation, genetic characterization, and virulence profiling of different Aeromonas species recovered from moribund hybrid catfish (Clarias spp.).

The high diversity of Aeromonas spp. results in various pathogenicity levels. This group of bacteria causes a serious disease named motile Aeromonas septicemia (MAS) in catfish (Clarias spp.). This study aimed to characterize the species and virulence gene diversity of Aeromonas spp. isolated from diseased catfish. Nine Aeromonas spp. were isolated from infected catfish cultivated in Java, Indonesia, and they were identified at the phenotypic and molecular levels (16S rDNA). The virulence genes assessed included aer/haem, alt, ast, flaA, lafA, and fstA. Phylogenetic analysis identified nine isolates of Aeromonas spp.: Aeromonas hydrophila (11.11%), Aeromonas caviae (11.11%), Aeromonas veronii bv. veronii (44.44%), and Aeromonas dhakensis (33.33%). Virulence genes, such as aer/haem, alt, ast, flaA, lafA, and fstA, were detected in all isolates at frequencies of approximately 100%, 66.67%, 88.89%, 100%, 55.56%, and 66.67%, respectively. This study is the first report on A. dhakensis recovered from an Indonesian catfish culture. Furthermore, our study revealed the presence of A. veronii bv veronii, a biovar that has not been reported before in Indonesia. This finding confirms that MAS was caused by multiple species of Aeromonas, notably A. dhakensis and A. veronii bv veronii, within Indonesian fish culture. The presence of these Aeromonas species with multiple virulence genes poses a significant threat to the freshwater aquaculture industry.

Immune protection and safety of Aeromonas veronii fliD deletion mutant as a live attenuated vaccine in Cyprinus carpio

Aeromonas veronii is one of the important pathogens causing motile Aeromonas septicemia (MAS) in aquaculture, leading to high fish mortality and severe economic losses. Live attenuated vaccines are considered to be an effective method to control bacterial infections and have been widely used in veterinary vaccines. In order to develop a safe and effective vaccine against MAS, the safety and protective efficacy of the fliD gene deletion strain of A. veronii as a live attenuated vaccine for Cyprinus carpio were evaluated in this study. After intraperitoneal injection vaccination, the A. veronii ΔfliD was able to colonize temporarily in the head kidney and spleen, and was finally cleared by C. carpio without causing clinical symptoms and affecting the growth performance of C. carpio. The significantly increased IgM levels in the serum indicated that both intraperitoneal injection and immersion vaccination induced a significant humoral immune response. The pro-inflammatory factors IL-1β, TNF-α and IL-6 were significantly increased at the early stage after intraperitoneal injection vaccination, indicating that A. veronii ΔfliD induced an inflammatory response in C. carpio, which facilitated bacterial clearance. In addition, a significant proliferation of head kidney lymphocytes was also observed after stimulation with A. veronii soluble antigen. The survival rates of C. carpio vaccinated by intraperitoneal injection and immersion were 83.33% and 56.67% after intraperitoneal challenge with wild-type A. veronii, compared with 13.33% and 16.67% in the control group. The histopathological examination showed that A. veronii ΔfliD could protect the liver and spleen against tissue lesion after challenge. These results indicate that attenuated strain A. veronii ΔfliD can induce an effective humoral and cellular immune response and provide protection against A. veronii for C. carpio without adverse effects, which can be developed as an effective live attenuated vaccine candidate to control the A. veronii infection.

First Report of Aeromonas veronii as an Emerging Bacterial Pathogen of Farmed Nile Tilapia (Oreochromis niloticus) in Brazil.

Brazil is one of the world's leading producers of Nile tilapia, Oreochromis niloticus. However, the industry faces a major challenge in terms of infectious diseases, as at least five new pathogens have been formally described in the last five years. Aeromonas species are Gram-negative anaerobic bacteria that are often described as fish pathogens causing Motile Aeromonas Septicemia (MAS). In late December 2022, an epidemic outbreak was reported in farmed Nile tilapia in the state of São Paulo, Brazil, characterized by clinical signs and gross pathology suggestive of MAS. The objective of this study was to isolate, identify, and characterize in vitro and in vivo the causative agent of this epidemic outbreak. The bacterial isolates were identified as Aeromonas veronii based on the homology of 16S rRNA (99.9%), gyrB (98.9%), and the rpoB gene (99.1%). A. veronii showed susceptibility only to florfenicol, while it was resistant to the other three antimicrobials tested, oxytetracycline, enrofloxacin, and amoxicillin. The lowest florfenicol concentration capable of inhibiting bacterial growth was ≤0.5 µg/mL. The phenotypic resistance of the A. veronii isolate observed for quinolones and tetracycline was genetically confirmed by the presence of the qnrS2 (colE plasmid) and tetA antibiotic-resistant genes, respectively. A. veronii isolate was highly pathogenic in juvenile Nile tilapia tested in vivo, showing a mortality rate ranging from 3 to 100% in the lowest (1.2 × 104) and highest (1.2 × 108) bacterial dose groups, respectively. To our knowledge, this study would constitute the first report of highly pathogenic and multidrug-resistant A. veronii associated with outbreaks and high mortality rates in tilapia farmed in commercial net cages in Brazil.

EFEKTIVITAS EKSTRAK BIJI PEPAYA (Carica papaya) TERHADAP KESEMBUHAN DAN KELANGSUNGAN HIDUP IKAN JELAWAT (Leptobarbus hoevenii) YANG DIINFEKSI BAKTERI Aeromonas hydrophila

Jelawat fish (Leptobarbus hoevenii) are found in several rivers in Kalimantan and Sumatra. Often encountered obstacles in fish farming is the bacterial disease Aeromonas hydrophila, known as Motile Aeromonas Septicemia (MAS). Treatment of disease can use natural ingredients that function as antimicrobials. Papaya seeds contain antimicrobials; besides that, they also contain secondary metabolites such as phenols, terpenoids, alkaloids, and saponins. The terpenoid group is the main component of papaya seeds and has antibacterial activity. This study aims to determine the effectiveness of the best papaya seed extract on the survival of jelawat fish infected with Aeromonas hydrophila bacteria. The treatment in this study used a completely randomized design (CRD) with 4 treatments and 3 replications. The treatments were treatment A (soaking with papaya seed extract at 0 ppm), treatment B (soaking with papaya seed extract at 40 ppm), treatment C (soaking with papaya seed extract at 80 ppm), and treatment D (soaking with papaya seed extract at 120 ppm). The results showed that the administration of papaya seed extract by immersion had a significant effect on the response to high feeding, with an average absolute weight growth value of 1.33 grams, a 100% survival rate, and wound healing in sharks infected with A. hydrophila bacteria starting to normalize or heal. Meanwhile, the water quality observed during the study was quite supportive for the survival of the jelawat, with temperatures ranging from 27–28, dissolved oxygen (DO) ranging from 7-8.7 mg/L, pH ranging from 6.5 to 7.3, and ammonia values from 0.1–0.3 mg/L. Giving 80 ppm through soaking papaya seed extract with jelawat fish is the best.

A review article effect of immersion Curcuma xanthorriza Roxb. extract for the survival of goldfish seed (Cyprinus carpio L.) after being infected with Aeromonas hydrophila

Long-term and inappropriate use of antibiotics can negatively impact goldfish seeds (Cyprinus carpio L.), which are affected by Motile Aeromonas Septicemia. One of the prevention efforts is the utilization of immunostimulants from natural ingredients such as Curcuma xanthorrhiza Roxb. Curcuma xanthorrhiza contains active substances in the form of curcuminoids, terpenoids, and essential oils that provide antibacterial activity with varying strength depending on the levels of components in the rhizome. This review article aims to provide scientific information regarding the effect of immersion time and the best concentration for the viability of goldfish seeds after being infected with Aeromonas hydrophila. The research method was experimental, using a factorial, Completely Randomized Design with several replications. Based on the results of the literature study that Curcuma xanthorriza Roxb. extract was known to utilization with a concentration of 0.6 g/l by injection of Aeromonas hydrophila 108cfu/mL gave the best effect on viability, which 100% absolute weight growth of 12.48 g/head and daily growth rate of 2.39%. The conclusion is a concentration of 0.6 g/l of Curcuma xanthorriza Roxb. extract had the best effect on viability so that it has the potential as an alternative natural antimicrobial.

Whole spectrum of Aeromonas hydrophila virulence determinants and the identification of novel SNPs using comparative pathogenomics

Aeromonas hydrophila is a ubiquitous fish pathogen and an opportunistic human pathogen. It is mostly found in aquatic habitats, but it has also been isolated from food and bottled mineral waters. It causes hemorrhagic septicemia, ulcerative disease, and motile Aeromonas septicemia (MAS) in fish and other aquatic animals. Moreover, it might cause gastroenteritis, wound infections, and septicemia in humans. Different variables influence A. hydrophila virulence, including the virulence genes expressed, host susceptibility, and environmental stresses. The identification of virulence factors for a bacterial pathogen will help in the development of preventive and control measures. 95 Aeromonas spp. genomes were examined in the current study, and 53 strains were determined to be valid A. hydrophila. These genomes were examined for pan- and core-genomes using a comparative genomics technique. A. hydrophila has an open pan-genome with 18,306 total genes and 1620 genes in its core-genome. In the pan-genome, 312 virulence genes have been detected. The effector delivery system category had the largest number of virulence genes (87), followed by immunological modulation and motility genes (69 and 46, respectively). This provides new insight into the pathogenicity of A. hydrophila. In the pan-genome, a few distinctive single-nucleotide polymorphisms (SNPs) have been identified in four genes, namely: d-glycero-beta-d-manno-heptose-1,7-bisphosphate 7-phosphatase, chemoreceptor glutamine deamidase, Spermidine N (1)-acetyltransferase, and maleylpyruvate isomerase, which are present in all A. hydrophila genomes, which make them molecular marker candidates for precise identification of A. hydrophila. Therefore, for precise diagnostic and discrimination results, we suggest these genes be considered when designing primers and probes for sequencing, multiplex-PCR, or real-time PCR.

Development of Bioluminescent Virulent Aeromonas hydrophila for Understanding Pathogenicity.

Virulent Aeromonas hydrophila (vAh) strains that cause motile Aeromonas septicemia (MAS) in farmed channel catfish (Ictalurus punctatus) have been an important problem for more than a decade. However, the routes of infection of vAh in catfish are not well understood. Therefore, it is critical to study the pathogenicity of vAh in catfish. To this goal, a new bioluminescence expression plasmid (pAKgfplux3) with the chloramphenicol acetyltransferase (cat) gene was constructed and mobilized into vAh strain ML09-119, yielding bioluminescent vAh (BvAh). After determining optimal chloramphenicol concentration, plasmid stability, bacteria number-bioluminescence relationship, and growth kinetics, the catfish were challenged with BvAh, and bioluminescent imaging (BLI) was conducted. Results showed that 5 to 10 µg/mL chloramphenicol was suitable for stable bioluminescence expression in vAh, with some growth reduction. In the absence of chloramphenicol, vAh could not maintain pAKgfplux3 stably, with the half-life being 16 h. Intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) challenges of catfish with BvAh and BLI showed that MAS progressed faster in the injection group, followed by the modified immersion and immersion groups. BvAh was detected around the anterior mouth, barbels, fin bases, fin epithelia, injured skin areas, and gills after experimental challenges. BLI revealed that skin breaks and gills are potential attachment and entry portals for vAh. Once vAh breaches the skin or epithelial surfaces, it can cause a systemic infection rapidly, spreading to all internal organs. To our best knowledge, this is the first study that reports the development of a bioluminescent vAh and provides visual evidence for catfish-vAh interactions. Findings are expected to provide a better understanding of vAh pathogenicity in catfish.

Innate Immune Response Assessment in Cyprinus carpio L. upon Experimental Administration with Artemia salina Bio-Encapsulated Aeromonas hydrophila Bacterin.

The present study aimed to analyze the enhancement of innate immune responses in juvenile-stage common carp (Cyprinus carpio L.), upon the administration of heat-killed Aeromonas hydrophila at a dosage of 1 × 107 CFU ml-1 through bio-encapsulation in the aquatic crustacean, Artemia salina. This work emphasizes the modulation of innate immune response when administered with the bio-encapsulated heat-killed antigen that acts as an inactivated vaccine against Motile Aeromonas Septicemia disease. Bio-encapsulated oral administration of antigens promotes innate immunity in juvenile-stage fishes. The optimization of effective bio-encapsulation of bacterin in Artemia salina nauplii was carried out and the best optimal conditions were chosen for immunization. The functional immune parameters such as myeloperoxidase, lysozyme, alkaline phosphatase, antiprotease and respiratory burst activity in serum, blood and intestinal tissue samples were analyzed along with blood differential leukocyte count and tissue histopathology studies. Both humoral and cellular immune responses analyzed were substantially induced or enhanced in the treatment groups in comparison with the control group. The results showed a significant variation in the bio-encapsulation group than the control group and also were comparable to the protection conferred with immersion route immunization under similar conditions. Thus, most of the innate non-specific immune responses are inducible, despite being constitutive of the fish immune system, to exhibit a basal level of protection and a road to better vaccination strategy in Cyprinus carpio L. aquaculture worldwide.

Proteomic analysis of liver tissue reveals Aeromonas hydrophila infection mediated modulation of host metabolic pathways in Labeo rohita

Aeromonas hydrophila (Ah) is a Gram-negative bacterium and a serious global pathogen causing Motile Aeromonas Septicaemia (MAS) in fish leading to global loss in aquaculture. Investigation of the molecular alterations of host tissues such as liver could be a powerful approach to identify mechanistic and diagnostic immune signatures of disease pathogenesis. We performed a proteomic analysis of Labeo rohita liver tissue to examine the protein dynamics in the host cells during Ah infection. The proteomic data was acquired using two strategies; discovery and targeted proteomics. Label-free quantification was performed between Control and challenged group (AH) to identify the differentially expressed proteins (DEPs). A total of 2525 proteins were identified and 157 were DEPs. DEPs include metabolic enzymes (CS, SUCLG2), antioxidative proteins, cytoskeletal proteins and immune related proteins (TLR3, CLEC4E). Pathways like lysosome pathway, apoptosis, metabolism of xenobiotics by cytochrome P450 were enriched by downregulated proteins. However, upregulated proteins majorly mapped to innate immune system, signaling of B cell receptor, proteosome pathway, ribosome, carbon metabolism and protein processing in ER. Our study would help in exploring the role of Toll-like receptors, C-type lectins and, metabolic intermediates like citrate and succinate in Ah pathogenesis to understand the Ah infection in fish. SignificanceBacterial diseases such as motile aeromonas septicaemia (MAS) are among the most serious problems in aquaculture industry. Small molecules that target the metabolism of the host have recently emerged as potential treatment possibilities in infectious diseases. However, the ability to develop new therapies is hampered due to lack of knowledge about pathogenesis mechanisms and host-pathogen interactions. We examined alterations in the host proteome during MAS caused by Aeromonas hydrophila (Ah) infection, in Labeo rohita liver tissue to find cellular proteins and processes affected by Ah infection. Upregulated proteins belong to innate immune system, signaling of B cell receptor, proteosome pathway, ribosome, carbon metabolism and protein processing. Our work is an important step towards leveraging host metabolism in targeting the disease by providing a bigger picture on proteome pathology correlation during Ah infection.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.

First evidence of extensively drug-resistant virulent Aeromonas dhakensis isolated from diseased endemic Mascara barb (Dawkinsia assimilis) in India

Aeromonas species is ubiquitously present in aquatic habitats, can cause motile Aeromonas septicemia (MAS). However, it is very complex to identify Aeromonas spp. based on its phenotypes. In the present study in mid-November 2018, we investigated a case of severe mortality of wild caught mascara barb (Dawkinsia assimilis) endemic to the Western Ghats, India. No cytopathic effect was observed in DRG and CSK fish cell line after inoculating filtrate of the tissue homogenates and found negative for all major viral pathogens. Here, aetiological agent was identified as bacteria, presumed as Aeromonas hydrophila, and Escherichia coli based on biochemical tests. Later, PCR assay was performed for the molecular confirmation by gyrB gene resulted in confirmation of Aeromonas as A. dhakensis with 99.63% similarity with A. dhakensis from SA1 of diseased pacu. Multiple antibiotic resistance (MAR) index of A. dhakensis was 0.9112. Further, A. dhakensis was positive for ten antibiotic resistance and seven virulence genes with β-hemolysis colonies. The LD50 value was found to be 3.42 × 105 CFU/mL in experimental trails. In the present study, a tetracycline, colistin, and ESBL resistant hypervirulent A. dhakensis was isolated from the diseased endemic mascara barb. Ten natural ingredients and five disinfectants found to be effective against A. dhakensis. To the best of our knowledge, this is the first evidence of extensively drug-resistant (XDR) virulent A. dhakensis associated with disease outbreak in endemic mascara barb in India.

Bacterial diseases of tilapia, their zoonotic potential and risk of antimicrobial resistance

AbstractTilapia culture is an important source of income and nutrition to many rural families. Since 2000, the production of tilapia increased and reached domestic and global markets. Major farmed species is Nile tilapia (Oreochromis niloticus), in earthen ponds and cage cultures. Intensification contributed to global tilapia disease outbreaks, with bacterial infections causing mortalities and morbidities, threatening sustainable production. At tilapia farms, high nutrient concentrations, water temperature and fish densities enhance bacterial growth including virulent bacterial clones and potential zoonotic bacteria. Global warming favours this. This review respectively provides a comprehensive overview of the most common and emerging bacterial pathogens, diseases, clinical presentations and diagnostics of tilapia, including bacteria and diseases with zoonotic potential. First, common bacterial disease outbreaks, including streptococcosis, motileAeromonassepticaemia, francisellosis, columnaris disease and vibriosis are described. Then, information on emerging bacterial infections of concern for tilapia, like edwardsiellosis throughEdwardsiella ictaluriandE.tarda, as well asAeromonas schubertiiis provided. Reports of infectious bacterial tilapia disease outbreaks from other bacteria, includingLactococcus garvieae,Aerococcus viridans,Pseudomonasspp.,Mycobacterium marinumandChlamydiaspp., and others are reviewed. Furthermore, bacteria with zoonotic potential, likeStreptococcus agalactiaeST283,S.iniae,Aeromonassp.,E.tarda,Vibrio vulnificuspathovar (pv)piscisandM.marinumare included in the review, to provide the most current overview of the disease risks affecting production and post‐harvest stages. Additionally, the status and risks of antimicrobial resistance in bacteria from tilapia and other cultured fish through imprudent use of antibiotics, and its future at a global level are provided.

Antimicrobial Activity of Excoecaria Agallocha Mangrove Extract in Inhibiting the Growth of Aeromonas Hydrophila by in-Vitro

One of the bacterial diseases that often attack freshwater fish is the red-sore disease, also known as aeromoniasis or Motile Aeromonas Septicemia (MAS). It is caused by bacteria from Aeromonas genus, such as Aeromonas hydrophila. A safe alternative treatment for pathogenic bacteria is the use of antibacterial compounds made from natural ingredients. The Excoecaria agallocha mangrove extract is one of potential alternatives that is useful as natural antimicrobials due to its various contents of antibacterial bioactive compounds. The objective of this study was to determine the antimicrobial activity of E. agallocha mangrove extract in inhibiting the growth of A. hydrophila. The research method was experimental with complete randomized design and factorial pattern of 3 factors (type of plant organ, bacterial strain, and concentration of extract), in 48 treatments and 3 replications. The solvents used consisted of methanol (polar) and n-hexane (non-polar). The plant organs used included leaves and stems. The bacterial strains consisted of GPl-04, GB-01, GL-01, GL-02, GJ-01, and GK-01 with concentrations of leaves and stems extracts of 0, 10, 20, and 30%. The parameter measured was the diameter of inhibition zone, which was done by using disc paper (Kirby Bauer method). Non-parametric Kruskall-Wallis Test and qualitative-descriptive data analysis were applied. The methanol extract of E. agallocha leaves in GJ-01 strain at 10 and 20% concentrations had antimicrobial activity with inhibition zone in 2.55 mm and 2.46 mm, respectively. The n-hexane extract of E. agallocha stem at 20 and 30% concentrations had antimicrobial activity with inhibition zones of 1.88 mm in GPl-04 strain and 1.58 mm in GB-01 strain, respectively. Extract of E. agallocha mangrove exhibited potential as a natural antibacterial to prevent aeromoniasis in fish.

Aeromonas hydrophila ST251 and Aeromonas dhakensis are major emerging pathogens of striped catfish in Vietnam.

Aeromonads are ubiquitous in aquatic environments and several species are opportunistic pathogens of fish. Disease losses caused by motile Aeromonas species, particularly Aeromonas hydrophila, can be challenging in intensive aquaculture, such as at striped catfish (Pangasianodon hypophthalmus) farms in Vietnam. Outbreaks require antibiotic treatments, but their application is undesirable due to risks posed by resistance. Vaccines are an attractive prophylactic and they must protect against the prevalent strains responsible for ongoing outbreaks. This present study aimed to characterize A. hydrophila strains associated with mortalities in striped catfish culture in the Mekong Delta by a polyphasic genotyping approach, with a view to developing more effective vaccines. During 2013-2019, 345 presumptive Aeromonas spp. isolates were collected at farms in eight provinces. Repetitive element sequence-based PCR, multi-locus sequence typing and whole-genome sequencing revealed most of the suspected 202 A. hydrophila isolates to belong to ST656 (n = 151), which corresponds to the closely-related species Aeromonas dhakensis, with a lesser proportion belonging to ST251 (n = 51), a hypervirulent lineage (vAh) of A. hydrophila already causing concern in global aquaculture. The A. dhakensis ST656 and vAh ST251 isolates from outbreaks possessed unique gene sets compared to published A. dhakensis and vAh ST251 genomes, including antibiotic-resistance genes. The sharing of resistance determinants to sulphonamides (sul1) and trimethoprim (dfrA1) suggests similar selection pressures acting on A. dhakensis ST656 and vAh ST251 lineages. The earliest isolate (a vAh ST251 from 2013) lacked most resistance genes, suggesting relatively recent acquisition and selection, and this underscores the need to reduce antibiotics use where possible to prolong their effectiveness. A novel PCR assay was designed and validated to distinguish A. dhakensis and vAh ST251 strains. This present study highlights for the first time A. dhakensis, a zoonotic species that can cause fatal human infection, to be an emerging pathogen in aquaculture in Vietnam, with widespread distribution in recent outbreaks of motile Aeromonas septicaemia in striped catfish. It also confirms vAh ST251 to have been present in the Mekong Delta since at least 2013. Appropriate isolates of A. dhakensis and vAh should be included in vaccines to prevent outbreaks and reduce the threat posed by antibiotic resistance.