Molecular Characterization of Ahp2, a Lytic Bacteriophage of Aeromonas hydrophila.
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ABSTRACT:
SUBMITTER: Wang JB
PROVIDER: S-EPMC8001559 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Phage therapy is an alternative approach to overcome the problem of multidrug resistance in bacteria. In this study, a bacteriophage named PZL-Ah152, which infects Aeromonas hydrophila, was isolated from sewage, and its biological characteristics and genome were studied. The genome contained 54 putative coding sequences and lacked known putative virulence factors, so it could be applied to phage therapy. Therefore, we performed a study to (i) investigate the efficacy of PZL-Ah152 in reducing the abundance of pathogenic A. hydrophila strain 152 in experimentally infected crucian carps, (ii) evaluate the safety of 12 consecutive days of intraperitoneal phage injection in crucian carps, and (iii) determine how bacteriophages impact the normal gut microbiota. The in vivo and in vitro results indicated that the phage could effectively eliminate A. hydrophila. Administering PZL-Ah152 (2 × 109 PFU) could effectively protect the fish (2 × 108 CFU/carp). Furthermore, a 12-day consecutive injection of PZL-Ah152 did not cause significant adverse effects in the main organs of the treated animals. We also found that members of the genus Aeromonas could enter and colonize the gut. The phage PZL-Ah152 reduced the number of colonies of the genus Aeromonas. However, no significant changes were observed in α-diversity and β-diversity parameters, which suggested that the consumed phage had little effect on the gut microbiota. All the results illustrated that PZL-Ah152 could be a new therapeutic method for infections caused by A. hydrophila.
| S-EPMC9315158 | biostudies-literature
Project description:Bacteriophages are increasingly being used as biological control agents against pathogenic bacteria. In the present study, we isolate and characterize bacteriophage Akh-2 from Geoje Island, South Korea, to evaluate its utility in controlling motile Aeromonas septicemia. Akh-2 lysed four of the seven Aeromonas hydrophila strains tested. Transmission electron microscopy analysis showed that Akh-2 belongs to the Siphoviridae family, with head and tail sizes of 50 ± 5 and 170 ± 5 nm, respectively. One-step growth curve analysis revealed that the phage has a latent period of 50 ± 5 min and a burst size of 139 ± 5 plaque-forming units per infected cell. The phage appeared stable in a pH range of 6-8 and a temperature range of -80 to 46 °C. Based on next-generation sequencing analysis, its genome is 114,901 bp in size, with a 44.22% G + C content and 254 open reading frames. During an artificial induction of the disease, loach (Misgurnus anguillicaudatus) treated with Akh-2 showed an increased survival rate and time compared with the non-treated control. Our results suggest that Akh-2 is a potential biological agent for the treatment of Aeromonas infections in fish.
| S-EPMC7157608 | biostudies-literature
Project description:Aeromonas hydrophila is an emerging foodborne pathogen causing human gastroenteritis. Aeromonas species isolated from food such as seafood presented multidrug-resistance (MDR), raising serious concerns regarding food safety and public health. The use of phages to infect bacteria is a defense against drug-resistant pathogens. In this study, phage ZPAH34 isolated from the lake sample exerted lytic activity against MDR A. hydrophila strain ZYAH75 and inhibited the biofilm on different food-contacting surfaces. ZPAH34 has a large dsDNA genome of 234 kb which belongs to a novel jumbo phage. However, its particle size is the smallest of known jumbo phages so far. Based on phylogenetic analysis, ZPAH34 was used to establish a new genus Chaoshanvirus. Biological characterization revealed that ZPAH34 exhibited wide environmental tolerance, and a high rapid adsorb and reproductive capacity. Food biocontrol experiments demonstrated that ZPAH34 reduces the viable count of A. hydrophila on fish fillets (2.31 log) and lettuce (3.28 log) with potential bactericidal effects. This study isolated and characterized jumbo phage ZPAH34 not only enriched the understanding of phage biological entity diversity and evolution because of its minimal virion size with large genome but also was the first usage of jumbo phage in food safety to eliminate A. hydrophila.
| S-EPMC10321303 | biostudies-literature
Project description:Aeromonas hydrophila is an opportunistic pathogen that frequently leads to significant mortality in various commercially cultured aquatic species. Bacteriophages offer an alternative strategy for pathogens elimination. In this study, we isolated, identified, and characterized a novel temperate A. hydrophila phage, designated as P05B. The bacteriophage P05B is a myovirus based on its morphological features, and possesses the capability to lyse A. hydrophila strains isolated from shrimp. The optimal multiplicity of infection (MOI), adsorption rate, latent period, and burst size for phage P05B were determined to be 0.001, 91.7 %, 20 min, and 483 PFU/cell, respectively. Phage P05B displayed stability across a range of temperatures (28-50 °C) and pH values (4.0-10.0). Sequence analysis unveiled that the genome of phage P05B comprises 32,302 base pairs with an average G + C content of 59.4 %. A total of 40 open reading frames (ORF) were encoded within the phage P05B genome. The comparative genomic analyses clearly implied that P05B might represent a novel species of the genus Bielevirus under Peduoviridae family. A phylogenetic tree was reconstructed, demonstrating that P05B shares a close evolutionary relationship with other Aeromonas and Aeromonas phages. In conclusion, this study increased our knowledge about a new temperate phage of A. hydrophila with strong lytic ability.
| S-EPMC10709362 | biostudies-literature
Project description:BackgroundAeromonas hydrophila is a prevalent pathogenic bacterium in aquaculture that causes economic loss around the world. Antimicrobials are used to control and prevent the incidence of bacterial pathogens in aquaculture. However, they lead to the emergence of antimicrobial resistance strains and the accumulation of antibiotic residues in fish tissue. To address these issues, bacteriophages may be promising alternatives to many antibiotics in combating bacterial infections in aquaculture.Materials and methodsThe phage specific to A. hydrophila was isolated from domestic wastewater. The morphology of phages was analyzed using transmission electron microscopy. The genomic DNA of the Aeromonas phage T65 strain (APT65) phage was sequenced with a paired-end read length of 2 × 150 bp. The genome sequence was assembled and annotated. The tRNAs were predicted, and antimicrobial resistance and virulence genes were screened. A representation of the APT65 genome was constructed.ResultsThe genome of APT65 is linear double-stranded DNA with 85188 base pairs having 116 open reading frames (ORFs) and a G + C content of 39.41%. The 32 ORFs were predicted to encode proteins with known phage functions. No virulence factors, antibiotic resistance genes, or temperate lifestyle genes were found. The phage is icosahedral and measures 60 nm in diameter. Based on the whole genome sequence, APT65 belongs to Lahexavirus.ConclusionsThe taxonomic analysis of the phage with a genome length of 85,188 bp revealed that it is a new species of the genus Lahexavirus. We announce the whole genome sequence of APT65, which should be named Lahexavirus APT65, as well as the absence of antimicrobial resistance and virulence factors from its genome. Based on our results, the Lahexavirus APT65 phage may have potential as a therapeutic agent to tackle antimicrobial resistance in aquaculture.
| S-EPMC10196078 | biostudies-literature
Project description:BackgroundAeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections.ResultsFive phages against pathogenic A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues.ConclusionsFive A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy.
| S-EPMC7268745 | biostudies-literature
Project description:BackgroundCombining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors.ResultsWe identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 μM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 μg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 μM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 μM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 μM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase.ConclusionsOur findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.
| S-EPMC5445283 | biostudies-literature
Project description:The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 μM, K(m)(actin) = 24 μM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 μM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.
| S-EPMC3481304 | biostudies-literature
Project description:Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.
| S-EPMC1489700 | biostudies-literature
Project description:Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.
| S-EPMC2430056 | biostudies-literature