Search

The PhoBR two-component system is a critical regulator of virulence in many bacterial pathogens, but its role in Aeromonas dhakensis remains poorly characterized. In this study, three markerless knockout mutants (Δ pho B, Δ pho R and Δ pho BR) were constructed to explore the function of PhoBR. Under low phosphate conditions (0.2 mM Pi), physiological analyses showed that all mutants exhibited defects in growth, motility, biofilm formation and resistance to acid, with Δ pho BR also showing resistance to H 2 O 2 , while no significant changes were observed in extracellular protease secretion. Cell adhesion and zebrafish infection assays indicated that PhoBR is crucial for A. dhakensis adhesion to EPC cells. The Δ pho BR mutant had the highest LD 50 value (1.77 × 107 CFU per fish), which was 38.15 times higher than that of the wild type strain. Additionally, Δ pho BR conferred the highest relative percent survival (RPS) of 61.5 % in zebrafish following two immunizations. These findings suggest that PhoBR positively regulates virulence in A. dhakensis and that the Δ pho BR mutant could serve as a potential vaccine candidate for the prevention of aeromoniasis in aquaculture. • Δ pho BR exhibits defective in growth, motility, biofilm formation, adhesion to EPC cells, and resistance to acid and H 2 O 2. • Δ pho BR is more attenuated in zebrafish than both Δ pho B and Δ pho R mutants. • Double vaccination with Δ pho BR confers 61.5 % relative percent survival (RPS) to zebrafish against the isogenic wild type strain. [ABSTRACT FROM AUTHOR]

As one of the most important economic fishes, largemouth bass (Micropterus salmoides) is being widely farmed around the world. With the improper expansion and high-density culture, largemouth bass is susceptible to an increasing number of pathogens. Particularly, co-infection of multiple pathogens is ubiquitous, which has become a main obstacle to the development of largemouth bass industry. In the study, a co-infected case caused by Aeromonas dhakensis (A. dhakensis) and Chryseobacterium indologenes (C. indologenes) in largemouth bass was firstly reported. Diseased fish with clinical signs such as exophthalmia, hemorrhage on gill cover, rot or ulceration on gill, skin and fin were collected for further diagnostics. Tissue lesions of liver, spleen and kidney were observed, such as necrosis, hemorrhage, vacuolization and disintegration. Two bacteria strains were isolated from liver, one appeared as a white or yellowish colony, and the other was characterized by golden yellow color. Based on the conjoint analysis of 16S rRNA and gyrB gene sequences, the isolated strains were identified as A. dhakensis and C. indologenes , respectively. Antibiotic sensitivity testing indicated that both A. dhakensis and C. indologenes were sensitive to flumequine, enrofloxacin, ceftazidime and minocycline, and resistant to penicillin, sulfamethoxine sodium, ampicillin and cefamezin. Largemouth bass showed similar clinical signs and tissue lesions to naturally diseased fish following co-infection with the isolated strains, indicating that the isolated strains fulfill Koch's postulates. Besides, a slightly increased pathogenicity was observed following co-infection compared with single infection. The data firstly reveal a co-infected case caused by A. dhakensis and C. indologenes in largemouth bass, not only enriching the relevant information of largemouth bass diseases, but also promoting the diagnosis, prevention and control of A. dhakensis and C. indologenes co-infection. • Co-infection of A. dhakensis and C. indologenes in largemouth bass is firstly reported. • Conjoint analysis of 16S rRNA and gyrB is performed to identify the isolated strains. • Both A. dhakensis and C. indologenes show sensitive to flumequine, enrofloxacin, ceftazidime and minocycline. • The isolated A. dhakensis and C. indologenes fulfill Koch's postulates. [ABSTRACT FROM AUTHOR]

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. • Aeromonas dhakensis were producing β-hemolysis colonies on 5% sheep blood agar and positive for seven virulence genes. • DNA gyrB gene sequencing of the isolates exhibited 99.63% similarity with A. dhakensis from SA1 of diseased pacu. • A. dhakensis was sensitive to only aminoglycosides with MAR index of 0.9112 with ten positive antibiotic resistance genes. • The LD50 value was found to be 3.42 × 105 CFU/mL in experimental trails and found to be highly virulent. • This study is the first evidence of extensively drug-resistant virulent A. dhakensis in endemic mascara barb in India [ABSTRACT FROM AUTHOR]

Aeromonas dhakensis is an emerging pathogen that is harmful to humans and aquaculture in tropical areas. The aim of this study was to understand the role of the two-component signal transduction system KdpDE in regulating the virulence of this species. A Δ kdp E mutant was generated via a markerless knockout. Phenotype analysis showed that the mutant was attenuated in growth, biofilm formation, mobility, ECPase activity, and resistance to H 2 O 2 and antimicrobial peptides under conditions of potassium depletion. A challenge experiment to analyze the virulence of A. dhakensis in zebrafish, via intraperitoneal injection, demonstrated that the clinicopathological signs of infection were dropsy, tail and/or fin bleeding, hepatonecrosis, spleen congestion, and nephrohemia. The LD 50 value of the mutant (9.02 × 106 CFU/ml) was 15.6-fold higher than that of the wild-type strain (5.77 × 105 CFU/ml). The survival rate of zebrafish was higher after double immunization (76.7%) than after single immunization with the mutant (36.7%). These data reveal for the first time that KdpDE is associated with the virulence of A. dhakensis. The Δ kdp E mutant is attenuated in virulence and shows high protective efficacy in adult zebrafish after double immunization, and is therefore a promising attenuated vaccine candidate. • dhakensis C160501 caused zebrafish hepatonecrosis, hyperaemia in skin, spleen and kidney. • △ kdp E attenuated A. dhakensis in biofilm formation, ECPase, mobility, as well as resistance to H 2 O 2 and AMPs of crocodile. • Δ kdp E resulted in the LD 50 of A. dhakensis increased 15.6-fold from 5.77 × 105 CFU/ml to 9.02 × 106 CFU/ml. • Double immunization of zebrafish with Δ kdp E mutant provided 76.7% of RPS against the challenge of isogenous C160501. [ABSTRACT FROM AUTHOR]