Hydrophila Strains Research Articles

Morin Protects Channel Catfish From Aeromonas hydrophila Infection by Blocking Aerolysin Activity.

Aeromonas hydrophila (A. hydrophila) is an opportunistic bacterial pathogen widely distributed in the environments, particular aquatic environment. The pathogen can cause a range of infections in both human and animals including fishes. However, the application of antibiotics in treatment of A. hydrophila infections leads to the emergence of resistant strains. Consequently, new approaches need to be developed in fighting this pathogen. Aerolysin, the chief virulence factor produced by pathogenic A. hydrophila strains has been employed as target identifying new drugs. In our present study, we found that morin, a flavonoid without anti-bacterial activity isolated from traditional Chinese medicine, could directly inhibit the hemolytic activity of aerolysin. To determine the binding sites and the action of mechanism of morin against AerA, several assays were performed. Ser36, Pro347, and Arg356 were identified as the main binding sites affecting the conformation of AerA and resulted in block of the heptameric formation. Moreover, morin could protect Vero cells from cell injury mediated by aerolysin. In vivo study showed that morin could provide a protection to channel catfish against A. hydrophila infection. These results demonstrated that morin could be developed as a promising candidate for the treatment of A. hydrophila infections by decreasing the pathogenesis of A. hydrophila.

Analysis of agglutinants elicited by antiserum of channel catfish immunized with extracellular proteins of virulent Aeromonas hydrophila

Motile Aeromonas septicemia (MAS), caused by new virulent Aeromonas hydrophila (vAh) strains, has been one of the major diseases in channel catfish in recent years. Previous studies showed that channel catfish developed immunity against vAh infection after immunization with the pathogen's extracellular proteins (ECP). To understand the mechanisms associated with the immunity, anti-ECP fish serum (antiserum) was analyzed in this study. Our results revealed that the antiserum elicited agglutination of both ECP and cells of vAh. Five fish proteins were identified in ECP agglutinants, including two innate immunity associated proteins (serotransferrin and rhamnose-binding lectin), two immunoglobulin M (IgM) molecules (IgM heavy chain and light chain) and a constitutively-produced protein (warm temperature acclimation protein). More than 68 vAh proteins in ECP were recognized and caused to aggregate by IgM in the antiserum. IgM was isolated from vAh cell agglutinants and the native IgM was shown to form a tetramer that was responsible for bacterial agglutination. Immunoblotting analysis indicated that the isolated native IgM was able to recognize some proteins in ECP, such as aerolysin and hemolysin (in the form of a high molecular weight heterologous polymer). Gene expression analysis by quantitative PCR showed that fish immunized with vAh ECP had more transcripts of genes coding for IgM, serotransferrin and rhamnose binding lectin than mock-immunized fish. Both innate and antibody-mediated immune responses in serum and expressed genes contributed to fish immunity upon immunization with ECP. Results of this study shed light on the versatility of vAh antigens and catfish IgM, which would help identify specific antigens for vaccine development and antigen specific antibodies in catfish.

Optimization and Partial Characterization Studies of Alkaline Protease from Aeromonas Hydrophila Strain YL 17 Isolated from Honey Sample

Optimization and Partial Characterization Studies of Alkaline Protease from Aeromonas Hydrophila Strain YL 17 Isolated from Honey Sample

Comparative genome analysis provides deep insights into Aeromonas hydrophila taxonomy and virulence-related factors

BackgroundAeromonas hydrophila is a potential zoonotic pathogen and primary fish pathogen. With overlapping characteristics, multiple isolates are often mislabelled and misclassified. Moreover, the potential pathogenic factors among the publicly available genomes in A. hydrophila strains of different origins have not yet been investigated.ResultsTo identify the valid strains of A. hydrophila and their pathogenic factors, we performed a pan-genomic study. It revealed that there were 13 mislabelled strains and 49 valid strains that were further verified by Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and in silico multiple locus strain typing (MLST). Multiple numbers of phages were detected among the strains and among them Aeromonas phi 018 was frequently present. The diversity in type III secretion system (T3SS) and conservation of type II and type VI secretion systems (T2SS and T6SS, respectively) among all the strains are important to study for designing future strategies. The most prevalent antibiotic resistances were found to be beta-lactamase, polymyxin and colistin resistances. The comparative analyses of sequence type (ST) 251 and other ST groups revealed that there were higher numbers of virulence factors in ST-251 than in other STs group.ConclusionPublicly available genomes have 13 mislabelled organisms, and there are only 49 valid A. hydrophila strains. This valid pan-genome identifies multiple prophages that can be further utilized. Different A. hydrophila strains harbour multiple virulence factors and antibiotic resistance genes. Identification of such factors is important for designing future treatment regimes.

Identifying genetic diversity of O antigens in Aeromonas hydrophila for molecular serotype detection.

Aeromonas hydrophila is a globally occurring, potentially virulent, gram-negative opportunistic pathogen that is known to cause water and food-borne diseases around the world. In this study, we use whole genome sequencing and in silico analyses to identify 14 putative O antigen gene clusters (OGCs) located downstream of the housekeeping genes acrB and/or oprM. We have also identified 7 novel OGCs by analyzing 15 publicly available genomes of different A. hydrophila strains. From the 14 OGCs identified initially, we have deduced that O antigen processing genes involved in the wzx/wzy pathway and the ABC transporter (wzm/wzt) pathway exhibit high molecular diversity among different A. hydrophila strains. Using these genes, we have developed a multiplexed Luminex-based array system that can identify up to 14 A. hydrophila strains. By combining our other results and including the sequences of processing genes from 13 other OGCs (7 OGCs identified from publicly available genome sequences and 6 OGCs that were previously published), we also have the data to create an array system that can identify 25 different A. hydrophila serotypes. Although clinical detection, epidemiological surveillance, and tracing of pathogenic bacteria are typically done using serotyping methods that rely on identifying bacterial surface O antigens through agglutination reactions with antisera, molecular methods such as the one we have developed may be quicker and more cost effective. Our assay shows high specificity, reproducibility, and sensitivity, being able to classify A. hydrophila strains using just 0.1 ng of genomic DNA. In conclusion, our findings indicate that a molecular serotyping system for A. hydrophila could be developed based on specific genes, providing an important molecular tool for the identification of A. hydrophila serotypes.

Aeromonas veronii and aerolysin are important for the pathogenesis of motile aeromonad septicemia in cyprinid fish.

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.

Multilocus sequence analysis revealed a high genotypic diversity of Aeromonas hydrophila infecting fish in Uganda.

A multilocus sequence analysis (MLSA) was carried out to delineate Aeromonas hydrophila from fish in Uganda. Five housekeeping genes including recA, gyrB, metG, gltA and pps; and the 16S rRNA gene were amplified and sequenced from a total of nine A.hydrophila isolates. The obtained sequences were edited, and consensus sequences generated for each gene locus. The housekeeping gene sequences were concatenated and phylogenetic analysis performed in MEGA version 7.0.2. Pairwise distances ranged from 0.000 to 0.118, highest within the gltA gene locus and lowest within the 16S rRNA gene. The average evolutionary diversity within isolates from the same source ranged between 0.002 and 0.037, and it was 0.033 between the different sources. Similar tree topologies were obtained from the different gene loci with recA, metG and gyrB being more consistent in discriminating isolates according to sources while the 16S rRNA gene had the lowest resolution. The concatenated tree had the highest discriminatory power. This study revealed that A.hydrophila strains infecting fish in Uganda are of diverse genotypes suggesting different sources of infection in a given outbreak. Efforts to minimize spread of the bacteria across sources should be emphasized to control infections of mixed genotypes.

The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.

Modulation of cytokine expression by dietary levan in the pathogen aggravated rohu, Labeo rohita fingerlings

A novel prebiotic, levan, has shown a positive beneficial role as a dietary immune-nutrient in fishes. However, the research investigations on the underlying mechanism of immune protection is unavailable, therefore in the present study we have attempted to reveal the effects of dietary microbial levan on the expression of cytokine genes in the pathogen challenged Labeo rohita fingerlings. Fish were randomly distributed in two treatment group in triplicates, control group fed without levan and treated group fed with 1.25% (w/w%) of microbial levan in the diet. After 60 days of feeding trial, fish from each group were challenged with virulent A. hydrophila strain and tissue samples were collected on different time points, 3 h, 6 h, 12 h, 24 h, 48 h, and 96 h. Dietary administration of microbial levan, significantly upregulated the m-RNA levels of pro-inflammatory cytokines like IL-1β, TNF-α and IL-12p40 in the intestine, gill, kidney and liver in a time dependent manner. An increase of 2.13-fold in the expression of IL-1β at 96 h post challenge in the kidney cells was detected, however at early time points (3 h & 6 h) expression of the same was down regulated. Upregulation of the TNF-α gene in the gill, liver and kidney at 6 h and in the intestine at 12 h was noticed. IL-12p40 m-RNA expressed highest upregulation of 3.65-fold at 3 h in the intestine and 2.0-fold at 12 h in the gill. IL-10 m-RNA expression of intestine in the infected L. rohita displayed 7.57-fold downregulation. Furthermore, increased respiratory burst activity and serum lysozyme activity was observed in a time dependent manner in infected rohu fed with levan supplementation. Overall, the current study revealed the upregulation of immune responsive cytokine genes (IL-1β, TNF-α and IL-12p40) with dietary microbial levan against the pathogen infected stress in Labeo rohita fingerlings and thus supports use of levan as food additive in aquaculture.

Development of a Colloidal Gold-Based Immunochromatographic Assay for the Rapid Detection of Edwardsiella Ictaluri.

Edwardsiella Ictaluri is known as the etiological agent of enteric septicaemia of channel catfish, causing heavy economic losses in the aquaculture industry. In this study, a colloidal gold-based immunochromatography assay (GICA) was developed for rapid detection of E. ictaluri. Briefly, monoclonal antibody (MAbs) and polyclonal antibody (PAbs) against E. ictaluri were prepared. Sensitivity of MAbs and PAbs to E. ictaluri was analyzed by Dot ELISA. Mouse MAb5D11 against E. ictaluri was conjugated with the 20 nm colloidal gold particles as the detector. Rabbit PAbs of E. ictaluri and goat anti-mouse IgG antibody was sprayed on nitrocellulose membranes as test line (T) and control line (C) respectively. The minimum detectable amount of this method to E. ictaluri was 5 × 106 CFU/mL. Cross reactions wouldn't occur when detecting E. tarda, Aeromonas hydrophila, V. parahaemolyticus and other several common standard strains. The result could be got in only 5 to 10 minutes. It didn't need professional technologies and testing experience. So this assay was very suitable for basic departments of aquatic product companies.

Isolation and Characterization of N-acyl Homoserine Lactone-Producing Bacteria From Cattle Rumen and Swine Intestines.

Quorum sensing systems regulate gene expression in response to bacterial population density. Acyl-homoserine lactones are a class of quorum sensing molecules found in cattle rumen that are thought to regulate the gene expression of enterohemorrhagic Escherichia coli and thus help this pathogen survive in animal gastrointestinal tracts. However, the specific bacteria that produce these signaling molecules in bovine and porcine gastrointestinal tracts are unknown. Here we developed methods to concentrate gastrointestinal fluids and screen the bacteria that produce acyl-homoserine lactones. We isolated a Pseudomonas aeruginosa strain YZ1 from cattle rumen, and an Aeromonas hydrophila strain YZ2 from pig intestine. Mass spectrometry analysis of culture supernatants indicated at least three specific classes of acyl-homoserine lactones produced by YZ1, and a C4-acyl-homoserine lactone produced by YZ2. Transformation of E. coli with P. aeruginosa or A. hydrophila luxI homologs,which can produce short- or long-chain acyl-homoserine lactones conferred upon E. coli the ability to synthesize acyl-homoserine lactones and affected gene expression, motility, and acid tolerance of E. coli. This is the first study reporting the isolation and characterization of acyl-homoserine lactone synthase-positive bacteria from cattle rumen and swine intestines.

Potential of Double-crested Cormorants ( Phalacrocorax auritus), American White Pelicans ( Pelecanus erythrorhynchos), and Wood Storks ( Mycteria americana) to Transmit a Hypervirulent Strain of Aeromonas hydrophila between Channel Catfish Culture Ponds.

Aeromonas hydrophila is a Gram-negative bacterium ubiquitous to freshwater and brackish aquatic environments that can cause disease in fish, humans, reptiles, and birds. Recent severe outbreaks of disease in commercial channel catfish ( Ictalurus punctatus) aquaculture ponds have been associated with a hypervirulent Aeromonas hydrophila strain (VAH) that is genetically distinct from less virulent strains. The epidemiology of this disease has not been determined. Given that research has shown that Great Egrets ( Ardea alba) can shed viable hypervirulent A. hydrophila after consuming diseased fish, we hypothesized that Double-crested Cormorants ( Phalacrocorax auritus), American White Pelicans ( Pelecanus erythrorhynchos), and Wood Storks ( Mycteria americana) could also serve as a reservoir for VAH and spread the pathogen during predation of fish in uninfected catfish ponds. All three species, when fed VAH-infected catfish, shed viable VAH in their feces, demonstrating their potential to spread VAH.

Mobile colistin resistance gene mcr-5 in porcine Aeromonas hydrophila.

ObjectivesTo characterize the mobile colistin resistance gene mcr-5 in Aeromonas hydrophila from backyard pigs in rural areas of China.MethodsPig faecal samples from 194 households were directly tested for the presence of mcr-5 by PCR assay and the phenotypic antimicrobial susceptibility profiles of the mcr-5-positive isolates were determined using the broth dilution method. The genomic location and transferability of mcr-5 were analysed by S1-PFGE with Southern blotting and DNA hybridization, and natural transformation, respectively. One strain isolated from an mcr-5-positive sample was subjected to WGS and the stability of the mcr-5-harbouring plasmid over successive generations was examined by subculturing.ResultsOne mcr-5-positive A. hydrophila isolate showing resistance, with a colistin MIC of 4 mg/L, was isolated from a backyard pig faecal sample. mcr-5 was located on a 7915 bp plasmid designated pI064-2, which could naturally transform into a colistin-susceptible A. hydrophila strain of porcine origin and mediated colistin resistance in both the original isolate and its transformants. The plasmid backbone (3790 bp) of pI064-2 showed 81% nucleotide sequence identity to the corresponding region of the ColE2-type plasmid pAsa1 from Aeromonas salmonicida, while similar replication primases are widely distributed among aeromonads, Enterobacteriaceae and Pseudomonas species.ConclusionsTo the best of our knowledge, this is the first identification of the novel colistin resistance gene mcr-5 in an A. hydrophila isolate from the faeces of a backyard pig. mcr-5 is expected to be able to disseminate among different bacterial species and genera.

Detection and analysis of hemolysin genes in Aeromonas hydrophila isolated from Gouramy (Osphronemus gouramy) by polymerase chain reaction (PCR)

The goal of this study was to detect of Aeromonas hydrophila carrying the hlyA gene in guramy by PCR assay. A total of 5 A. hydrophila strains were isolated from gouramy with different location and furthermore genotypic of all A. hydrophila strains havedetected by PCR assay for 16S rRNA gene. The primers used in the PCR targeted a 592-bp fragment of the hlyA gene coding for the hemolysin gene. Particularly hlyA genes are responsible for haemolysin toxins production in this genus. After gel electrophoresis, the amplicons from representative strains of the A. hydrophila were purified using extraction kit and were subjected to the DNA sequencing analysis. The results showed that: (i) the 592bp amplicon of the hlyA gene was detected in 5/6 of the A. hydrophila; (ii) the nucleotide blast results of hemolysin gene sequences of the strains of A. hydrophila revealed a high homology of 90-97 % with published sequences, and;(iii) the protein blast showed 95-98 % homology when compared to the published sequences. The PCR clearly identified the haemolysin-producing strains of A. hydrophila by detection in hlyA genes and may have application as a rapid species-specific virulence test.

Characterization and genomic analyses of Aeromonas hydrophila phages AhSzq-1 and AhSzw-1, isolates representing new species within the T5virus genus.

In this study, two bacteriophage isolates, AhSzq-1 and AhSzw-1 that specifically infect Aeromonas hydrophila strain KT998822, were isolated from seawater and characterized. One-step growth curves showed that the latent period of AhSzq-1 and AhSzw-1 are 50 min and 60 min, respectively. The sequence similarities between AhSzq-1 and AhSzw-1 were 88% at the DNA and 83% at the protein level, suggesting that these two phages are representatives of two different species. The virion morphology, DNA genome size and terminal repeats of these two phages are similar to those of viruses classified as T5virus phages. Both phylogenetic analyses and proteomic comparison show that AhSzq-1 and AhSzw-1 group with members of the T5virus genus. We thus propose these two phages as representative isolates of two new species within the T5virus genus.

Molecular characteristics and virulence analysis of eight Aeromonas hydrophila isolates obtained from diseased Amur sturgeon Acipenser schrenckii Brandt, 1869.

Aeromonas hydrophila is an opportunistic pathogen of a variety of aquatic animals that displays extreme diversity in drug resistance, phenotypes, virulence genes, and virulence. In this study, eight pathogenic A. hydrophila strains were isolated from diseased Amur sturgeons and investigated for their sensitivity to select antibiotics, their phenotype, virulence genes, and virulence. According to the phylogenetic analysis of the DNA gyrase subunit B protein, the eight isolates formed a single branch in the A. hydrophila group. The antibiotics ceftazidime, cefuroxime, cefoperazone, cefotaxime, ceftriaxone, aztreonam, and cefepime appeared effective against them. All of the isolates possessed the virulence genes for aerolysin, flagellin, heat-stable cytotonic enterotoxin, heat-labile cytotonic enterotoxin, hemolysin, and elastase, while only one isolate, HZ8, possessed the gene for lateral flagella. The cytolytic enterotoxin and lipase genes were present in all isolates, except in ZJ10 and ZJ12. Enterobacterial repetitive intergenic consensus sequence PCR indicated that the eight A. hydrophila isolates could be divided into four types. Isolates YW2, TR3, HZ8 and ZJ10, each representing a different type, were selected for challenge experiments. The challenge tests revealed that isolate HZ8 had the lowest lethal dose, causing 50% mortality at 2.30 × 104 colony forming units (cfu)/ml. The isolate ZJ10 had the highest LD50, 1.25 × 106 cfu/ml. Knowledge of the characteristics of the A. hydrophila isolates obtained from Amur sturgeon will be beneficial in developing potential disease control strategies.

Detection of some virulence genes in A. hydrophila and A. caviae isolated from fresh water fishes at Qalubia Governorate

The study was conducted on 225 diseased fish samples, 125 Nile tilapia (Oreochromis niloticus)and 100 Catfish(Claris gariepinus) , collected from different fish markets at Qalubia Governorateduring the period from January (2016) to May (2017) for detection of Aeromonas species. Thesamples were taken from apparently pathognomonic lesions in muscle, kidney, liver, intestine andspleen after clinical and postmortem examination for bacteriological examination. The resultsrevealed that, 125 Aeromonas species were isolated from the examined samples where A.hydrophila and A. caviae were identified. Accurately 114 (91.2 %) A. hydrophila strains, 63(50.4%) and 51 (40.8%) were isolated from C. gariepinus and O. niloticus fishes respectively.Meanwhile, 11(8.8 %) A. caviae strains, 7 (5.6%) and 4 (3.2%) from C. garicpinus and O. niloticusfishes respectively. Further PCR results for virulence genes in isolated Aeromonas strains indicatedthat, aero gene was detected in 9 out of 10 A .hydrophila studied strains and in 3 out of 6 A. caviae ;hly gene in 7 out of 10 A .hydrophila and in 2 out of 6 A. caviae; Ahcytoen gene in 6 out of 10 A.hydrophila and in 1 out of 6 A. caviae ; act gene in 6 out of 10 A .hydrophila and in 3 out of 6 A.caviae and ast gene in 7 out of 10 A .hydrophila and in 3 out of 6 A. caviae studied strains. Finally,the production of a wide array of virulence factors by isolated strains is indicative of their potentialto cause diseases in fishes and humans.

Draft genome sequence of a multidrug-resistant Aeromonas hydrophila ST508 strain carrying rmtD and blaCTX-M-131 isolated from a bloodstream infection.

Here we report the draft genome sequence of a multidrug-resistant (MDR) Aeromonas hydrophila strain belonging to sequence type 508 (ST508) isolated from a human bloodstream infection. Assembly and annotation of this draft genome resulted in 5028498bp and revealed the presence of 16S rRNA methylase rmtD and blaCTX-M-131 genes encoding high-level resistance to aminoglycosides and cephalosporins, respectively, as well as multiple virulence genes. This draft genome can provide significant information for understanding mechanisms on the establishment and treatment of infections caused by this pathogen.

Draft Genome Sequence of Aeromonas hydrophila Strain BSK-10 (Serotype O97), Isolated from Carassius carassius with Motile Aeromonad Septicemia in China.

ABSTRACT We report here a draft genome sequence of Aeromonas hydrophila strain BSK-10, belonging to serotype O97, isolated from crucian carp (Carassius carassius) with motile aeromonad septicemia in Zhejiang, China. The assembly resulted in 34 scaffolds totaling approximately 4.97 Mb, with an average G+C content of 60.97% and 4,594 predicted coding genes.

Pathogenicity of Aeromonas hydrophila isolated from diseased sharp teeth catfish, Clarias gariepinus, with special reference to the lethality of its extracellular products

0px; > A study was conducted to investigate the pathogenicity and the median lethal dose (LD50) of Aeromonas hydrophila isolated from clinically diseased catfish against apparently healthy homologous fish to evaluate the lethality of extra-cellular products (ECPs) of the isolated strain in vivo. For pathogenicity experiment, five different concentrations of Aeromonas hydrophila strain BNS 01614 including 3× 108, 1.5 × 108, 1.5 × 107, 1.5 × 106 and 1.5 × 105 CFU/fish used via intra peritoneal. The results revealed that pathogenicity of BNS 01614 was confirmed by the mortality of 30 % to 100 % of all tested fish within 4 to 12 days with LD50 1.5 × 107 CFU/fish. The Concentrated extracellular products (ECPs) of the selected bacterium were prepared and confirmed to be toxic in Clarias garipineus with LC50 of 20µg.