Cytotonic Enterotoxin Research Articles

Analysis of global Aeromonas caviae genomes revealed that strains carrying T6SS are more common in human gastroenteritis than in environmental sources and are often phylogenetically related.

Aeromonas caviae is an emerging human enteric pathogen. However, the genomic features and virulence genes of A. caviae strains from human gastroenteritis and other sources have not been fully elucidated. Here, we conducted a genomic analysis of 565 global A. caviae strains isolated from different sources, including 261 strains isolated from faecal samples of gastroenteritis patients, of which 18 genomes were sequenced in this study. The presence of bacterial virulence genes and secretion systems in A. caviae strains from different sources was compared, and the phylogenetic relationship of A. caviae strains was assessed based on the core genome. The complete genome of A. caviae strain A20-9 isolated from a gastroenteritis patient was obtained in this study, from which 300 putative virulence factors and a T4SS-encoding plasmid, pAC, were identified. Genes encoding T4SS were also identified in a novel genomic island, ACI-1, from other T4SS-positive strains. The prevalence of T4SS was significantly lower in A. caviae strains from gastroenteritis patients than in environmental strains (3 %, P<0.0001 vs 14 %, P<0.01). Conversely, the prevalence of T6SS was significantly higher in A. caviae strains isolated from gastroenteritis patients than in environmental strains (25 %, P<0.05 vs 13 %, P<0.01). Four phylogenetic clusters were formed based on the core genome of 565 A. caviae strains, and strains carrying T6SS often showed close phylogenetic relationships. T3SS, aerolysin and thermostable cytotonic enterotoxin were absent in all 565 A. caviae strains. Our findings provide novel information on the genomic features of A. caviae and suggest that T6SS may play a role in A. caviae-induced human gastroenteritis.

Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles.

Aeromonas jandaei is a gram-negative bacterium commonly found in aquatic environments and can induce illnesses in amphibians, reptiles and aquatic animals. In this study, a strain of bacteria was isolated from the diseased Chinese soft-shell turtle (Pelodiscus sinensis), then named strain JDP-FX. This isolate was identified as A. jandaei after analysis of morphological, physiological and biochemical characteristics, as well as 16S rRNA and gyrB gene sequences. Virulence genetic testing further detected temperature-sensitive protease (eprCAI), type III secretion system (TTSS) (ascv), nuclease (nuc), cytotonic enterotoxin (alt) and serine proteinase (ser) in JDP-FX. Compared with healthy Chinese soft-shell turtle, the serum levels of total protein (TP), albumin (ALB) and globulin (GLB) were significantly decreased in the diseased Chinese soft-shell turtle, while, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly increased. Histopathological observations showed that multiple tissues, including intestinal mucosa, liver and kidney, were severely damaged in the diseased Chinese soft-shell turtle. Moreover, the diseased Chinese soft-shell turtle had significant cell degeneration, necrosis, sloughing and interstitial inflammatory cell infiltration. The pathogenicity of JDP-FX was tested via artificial infection. The median lethal dosage (LD50 ) of the strain was 1.05 × 105 colony forming units (CFU/g) per weight of Chinese soft-shell turtle. Drug susceptibility analysis revealed that JDP-FX was susceptible to ceftazidime, minocycline, cefoperazone, ceftriaxone and piperacillin. In addition, JDP-FX was resistant to doxycycline, florfenicol, sulfonamides, gentamicin, ampicillin and neomycin. Therefore, this study may provide guidance for further research into the diagnosis, prevention and treatment of JDP-FX infection.

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.

Detection of Virulence Genes, Antimicrobial Susceptibility and Pathogenicity of Aeromonas veronii Isolates from Labeo rohita and Catla catla

Background: Aeromonas veronii is the most common bacterium responsible for diseases in freshwater fish rearing systems. Multiple factors can be involved in the virulence processes of Aeromonas bacteria. Hence, the purpose of the present investigation was to evaluate the virulence genes, antimicrobial susceptibility and pathogenicity of A. veronii isolated from freshwater fishes. Methods: In this investigation, we isolated A. veronii from cultured freshwater fishes, Labeo rohita and Catla catla. A. veronii was identified by bacterial staining and culture characteristics. In addition, Polymerase chain reaction (PCR) was used to evaluate the distribution of nine virulence genes including aerolysin, cytotonic enterotoxin (442, 272 bp), elastase, enolase, flagellin, lipase, serine protease and DNase. Result: A total of 88 A. veronii strains were isolated, which includes 56 strains from rohu and 33 strains from catla. The strains were Gram-negative, rod-shaped, motile bacteria and the colonies are yellow on blood agar. All the A. veronii strains were positive for at least one or more virulence genes tested. The isolates carried more virulence genes, especially in the combination of aer,alt, ela, lip, AhyB genes were found to be more virulent. Antimicrobial susceptibility to 17 antibiotics was determined and the strains of A. veronii showed 100% resistance to tetracycline, pencillin and â-lactum group of antibiotics. In addition, multiple antibiotic resistance (MAR) indexes ranged from 0.24 to 0.76, suggesting that they originated from high risk contaminated zones were (W. Godavari and Nellore districts) antimicrobials are often used.

Multidrug-resistant Aeromonas bacteria prevalence in Nile tilapia broodstock

BackgroundAeromonas 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.ResultsSequencing 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 × 104 to 3.02 × 106 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 (TiO2 NPs) with three sizes (13, 32, and 123 nm). If A. hydrophila strains with a high MDR index were tested against TiO2 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 TiO2 NP treatment.ConclusionsNile tilapia broodstock, mortality is associated with different A. hydrophila strains, which harbored virulent and MDR genes. Furthermore, TiO2 NPs had bactericidal activity, thereby resulting in a considerable reduction in bacterial load.

Isolation, identification, and characterization of pathogenic Aeromonas hydrophila from critically endangered Acipenser baerii

The Siberian sturgeon (Acipenser baerii) is a long-lived and late-maturing fish; its natural populations are considered endangered and listed in the first class of protected animals in Kazakhstan. To date, attempts have been made to increase the number of sturgeons through artificial reproduction in aquaculture and a release of the resultant juveniles into the Ural River, which flows into the Caspian Sea. This species faces an increased risk of Aeromonas infections. Aeromonas hydrophila is the most important sturgeon pathogen in Kazakhstan, but studies on A. hydrophila infection in Kazakhstan are still inconclusive. In the present work, our purpose was to isolate and characterize dominant bacteria in diseased A. baerii. This isolate, tentatively named AB005, was identified as A. hydrophila in an analysis of its morphological, physiological, and biochemical features and 16S ribosomal-RNA and gyrB gene sequences. A pathogenicity test was carried out for the isolate on healthy Oreochromis niloticus and Acipenser ruthenus via intraperitoneal injection along the caudal peduncle of the fish. Half-lethal doses (LD50) of isolate AB005 for O. niloticus and A. ruthenus were determined: 8.37 × 105 and 2.89 × 106 colony-forming units per milliliter, respectively. Virulence gene profiling revealed the presence of seven virulence genes related to pathogenicity (acyltransferase, phospholipase A, serine protease, heat-stable cytotonic enterotoxin, nuclease, and aerolysins A and B) in this A. hydrophila isolate. Drug sensitivity testing showed that the isolate is sensitive to quinolones, aminoglycosides, nitrofurans, amphenicols, and tetracyclines. The present findings will lay the foundation for future research on this pathogen in Siberian-sturgeon aquaculture.

Prevalence, Virulence Gene Distribution and Alarming the Multidrug Resistance of Aeromonas hydrophila Associated with Disease Outbreaks in Freshwater Aquaculture.

The study aims to evaluate the infection prevalence, virulence gene distribution and antimicrobial resistance of Aeromonas hydrophila associated in diseased outbreaks of cultured freshwater fish in Northern Vietnam. The confirmed A. hydrophila were screened for the presence of the five pitutative-virulence genes including aerolysin (aerA), hemolysin (hlyA), cytotonic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), and heat-stable enterotoxin (ast), and examined the susceptibility to 16 antibiotics. A total of 236 A. hydrophila isolates were recovered and confirmed from 506 diseased fish by phenotypic tests, PCR assays, and gyrB, rpoB sequenced analyses, corresponding to the infection prevalence at 46.4%. A total of 88.9% of A. hydrophila isolates harbored at least one of the tested virulence genes. The genes aerA and act were most frequently found (80.5% and 80.1%, respectively) while the ast gene was absent in all isolates. The resistance to oxacillin, amoxicillin and vancomycin exhibited the highest frequencies (>70%), followed by erythromycin, oxytetracycline, florfenicol, and sulfamethoxazole/trimethoprim (9.3–47.2%). The multiple antibiotic resistance (MAR) index ranged between 0.13–0.88 with 74.7% of the isolates having MAR values higher than 0.2. The results present a warning for aquaculture farmers and managers in preventing the spread of A. hydrophila and minimizing antibiotic resistance of this pathogen in fish farming systems.

Biological characteristics of enteropathogenic Aeromonas species isolated from different Egyptian foods

A total of 185 food samples in Sharkia Governorate, Egypt were analyzed for the presence of Aeromonas spp. The recovered isolates were characterized by biochemical tests based on Bergey’s Manual of Systematic Bacteriology and confirmed by API-20E kit. Of the samples analyzed, 56 (30.27 %) were positive for Aeromonas spp., with the highest incidence in raw milk (55 %) followed by Nile tilapia (48.6 %). A. hydrophila and A. salmonicida were the most predominantly isolated from raw milk and A. caviae was the most prevalent in raw lean beef. A variety of extracellular virulence factors were determined and 83.3 % exhibited β-hemolytic activity; 80 % exhibited lecithinase activity; 75 % exhibited protease activity and 90 % exhibited lipolytic activity. Furthermore, the cytotoxic (act) and the cytotonic (alt) enterotoxins were screened in three selected isolates using PCR assay and the data showed the detection of the two genes with predicted sizes of 232 bp and 442 bp fragments, respectively. These findings indicated a major health risk of infection with enterotoxigenic Aeromonas bacteria for people consuming raw and improper cooked food in Egypt.

Virulence potential of Aeromonas hydrophila isolated from apparently healthy freshwater food fish

Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90–98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah+act+ alt+ascV+eno+lip+ast+ on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD50 for 9C strain was 6.81 × 104 CFU/mL and 7.62 × 105 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.

Prevalence and virulence characteristics of Aeromonas species isolated from fish farms in Egypt

Background/Objective: Fish is one of the most valuable food for human consumption. However, fish may also act as a source of foodborne pathogens including Aeromonas species which caused a serious threat to a public health concern. This study aimed to investigate the prevalence and virulence characteristics of Aeromonas species isolated from fish farms in Egypt. Methods: A total of 100 random samples of freshwater fish represented by Nile tilapia and Mugil cephalus (50 of each) were collected from different fish farms in Kafr El-Sheikh governorate and examined bacteriologically and biochemically for the presence of Aeromonas species. Multiplex PCR was done to detect some virulence-associated genes in Aeromonas hydrophila isolates. Results: The obtained results revealed that the incidence of Aeromonas species in Nile tilapia and Mugil cephalus were 32 (64%) and 25 (50%), respectively. The most prevalent Aeromonas species isolated from Nile tilapia were Aeromonas caviae 13 (40.6%), Aeromonas hydrophila 8 (25%), Aeromonas sobria 7 (21.9%), Aeromonas veronii 3 (9.4%), and Aeromonas fluvialis 1 (3.1%), while from fresh Mugil cephalus were Aeromonas sobria 11 (44%), Aeromonas caviae 7 (28%), Aeromonas hydrophila 5 (20%), and Aeromonas veronii 2 (8%). The isolated Aeromonas hydrophila concealed some virulence genes responsible for their pathogenicities such as aerolysin gene (aerA) and cytotonic enterotoxin gene (altA). Conclusion: The hygienic measures should be applied to prevent fish contamination with Aeromonas species.

Aeromonas veronii associated with red gill disease and its induced immune response in Macrobrachium nipponense

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. In this study, the pathogenic of A. veronii was isolated from diseased Macrobrachium nipponense showing signs of weakness, poor appetite and red gills in some farms in Jiangsu province, China. The pathogenicity of A. veronii to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. veronii NJ1 was pathogenic to M. nipponense, and the LD50 was 4.5 × 106 CFU/ml at 7 d post infection. Detection virulence factors of the A. veronii NJ1 showed that A. veronii NJ1 produced caseinase, lipase, amylase, lecithinase, gelatinase and haemolysin. Detection of virulence-related genes showed that A. veronii NJ1 was positive for aerolysin (aer), outer membrane protein (ompA), adhesin (aha), flagellin (flaA, flgH, flgM, flgN), lipoidase (lip), cytotonic enterotoxins (act, alt, ast), serine protease (ser), DNases (exu), haemolysin (hlyA), type IV pilus (tapA) and fimbrillin (fim). Antibiotic sensitivity testing indicated A. veronii NJ1 was susceptible to quinolones, but resistant to penicillins. Additionally, the immune-related gene expression in M. nipponense was evaluated, and the results showed that anti-lipopolysaccharide factor (ALF1, ALF2, ALF3), Cusrin1, Cusrin2, Cusrin3, Haemocyanin, i-lysozyme and Prophenoloxidase were significantly upregulated from 6 to 24 hr after A. veronii infection. The results of our study suggested that A. veronii was an aetiological element in the mass mortalities of M. nipponense and this study provided preliminary insights into the diversity in the immune response of M. nipponense to the A. veronii infection.

Virulence-Associated Genes and Antimicrobial Resistance of Aeromonas hydrophila Isolates from Animal, Food, and Human Sources in Brazil.

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.

Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassius auratus gibelio).

Aeromonas species often cause disease in farmed fish. In the present study, dominant bacteria were isolated from diseased crucian carp (Carassius auratus gibelio). Based on this, a bacterial isolate was tentatively named CFJY-623. This isolate was identified as Aeromonas veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Six virulence genes related to pathogenicity including aerolysin, cytotonic enterotoxins, elastase, glycerophospholipid: cholesterol acyltransferase, lipase, and serine protease were identified in this A. veronii isolate. The median lethal dosage (LD50) of the CFJY-623 isolate for crucian carp was determined as 1.31 × 107 CFU/mL. Artificial experimental infection showed that the CFJY-623 isolate caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Drug sensitivity testing showed that the isolate was susceptible to aminoglycosides, carbapenemes, and nitrofurans. Exploring its growing features showed that this isolate exhibited a high level of environmental adaptability. These results provided a scientific basis for the identification of A. veronii and treatment for fish infected by this pathogen.

MOLECULAR IDENTIFICATION OF Aeromonas hydrophila STRAINS RECOVERED FROM KAFRELSHEIKH FISH FARMS

The aim of this study was to recover Aeromonas hydrophila from Kafrelsheikh tilapia (Oreochromis niloticus) farms and to study its virulence. Adult fish (n = 100) exhibiting the onset of clinical signs were bacteriologically examined using aeromonas isolation base medium. The isolates were identified by PCR using A. hydrophila-specific 16S rRNA primers and the virulence was determined using specific primers for ten virulence genes targeting hidrolipase (Lip), elastase (ahyB), lipase (pla/lip), aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature sensitive protease (eprCAI), serine protease (Ahp), haemolysin (hlyA), and cytotonic heat stable enterotoxin (ast). Molecular screening revealed presence of 35 isolates positive for A. hydrophila-specific 16S rRNA and 4 virulence genes (aer, pla/lip, ast, and hlyA). It also showed that the majority of the examined strains carried one or more virulence genes. These data indicate higher virulence for A. hydrophila in infected O. niloticus in Kafrelsheikh fish farms.Key words: PCR; A. hydrophila; virulence genes

Insecticidal Toxicity of Yersinia frederiksenii Involves the Novel Enterotoxin YacT.

The genus Yersinia comprises 19 species of which three are known as human and animal pathogens. Some species display toxicity toward invertebrates using the so-called toxin complex (TC) and/or determinants that are not yet known. Recent studies showed a remarkable variability of insecticidal activities when representatives of different Yersinia species (spp.) were subcutaneously injected into the greater wax moth, Galleria mellonella. Here, we demonstrate that Y. intermedia and Y. frederiksenii are highly toxic to this insect. A member of Y. Enterocolitica phylogroup 1B killed G. mellonella larvae with injection doses of approximately 38 cells only, thus resembling the insecticidal activity of Photorhabdus luminescens. The pathogenicity Yersinia spp. displays toward the larvae was higher at 15°C than at 30°C and independent of the TC. However, upon subtraction of all genes of the low-pathogenic Y. enterocolitica strain W22703 from the genomes of Y. intermedia and Y. frederiksenii, we identified a set of genes that may be responsible for the toxicity of these two species. Indeed, a mutant of Y. frederiksenii lacking yacT, a gene that encodes a protein similar to the heat-stable cytotonic enterotoxin (Ast) of Aeromonas hydrophila, exhibited a reduced pathogenicity toward G. mellonella larvae and altered the morphology of hemocytes. The data suggests that the repertoire of virulence determinants present in environmental Yersinia species remains to be elucidated.

Genotypic detection of some virulence factors among Aeromonas hydrophila Isolated from Diarrhea Cases (Iraq)

The present study included the detection ofsome virulence factorsof Aeromonas hydrophila under molecular level to clinical isolates were taken from patients suffering from diarrhea during the period from July (2017) to October (2017). Molecular detection of Hemolysin gene (ahh) was done for all isolates. The results showed that all isolates (100%) gave positive results for this virulence gene. the positive results were detected by the presence of (130) bp bands when compared with allelic ladder. The genomic DNA of the samples was extracted and bands were observed by performing agarose gel electrophoresis. When PCR was performed,results clearly indicate that all isolated organisms contained serine protease gene and all the amplified products produced a band at the level of (900 bp) when compared with the allelic ladder. Molecular detection of this gene was carried out by using a specific PCR primer were done by comparison with allelic ladder which gave a (309bp) It was found that (Aerolysin) gene present in (12) (75%) of the positive samples. Lip gene was also detected in A. hydrophila samples and found that all 16 samples (100%) gave positive results to this gene which gave molecular length (382) bp. Molecular study was carried out to show the sequence identity of cytotonic enterotoxins gene in Aeromonas spp. to that in A. hydrophila. Analysis of the A. hydrophila genome revealed a number of a putative virulence factors,including a gene that heat-labile cytotonic enterotoxin (alt). Our study showed that all (16) isolates (100%) gave positive results to this gene,which gave molecular length (442)bp. Molecular detection of cytotonic enterotoxins gene (ast) was done for (16) A. hydrophila isolates and the results showed that all isolates have this gene (100%). The positive results for (ast) virulence were detected by the presence of (331) bp band compared with allelic ladder.

Genotypic Detection of Some Virulence Factors Among Aeromonas hydrophila Isolated from Diarrhea Cases (Iraq)

The present study included the detection ofsome virulence factorsof Aeromonas hydrophila under molecular level to clinical isolates were taken from patients suffering from diarrhea during the period from July (2017) to October (2017). Molecular detection of Hemolysin gene (ahh) was done for all isolates. The results showed that all isolates (100%) gave positive results for this virulence gene. the positive results were detected by the presence of (130) bp bands when compared with allelic ladder. The genomic DNA of the samples was extracted and bands were observed by performing agarose gel electrophoresis. When PCR was performed,results clearly indicate that all isolated organisms contained serine protease gene and all the amplified products produced a band at the level of (900 bp) when compared with the allelic ladder. Molecular detection of this gene was carried out by using a specific PCR primer were done by comparison with allelic ladder which gave a (309bp) It was found that (Aerolysin) gene present in (12) (75%) of the positive samples. Lip gene was also detected in A. hydrophila samples and found that all 16 samples (100%) gave positive results to this gene which gave molecular length (382) bp. Molecular study was carried out to show the sequence identity of cytotonic enterotoxins gene in Aeromonas spp. to that in A. hydrophila. Analysis of the A. hydrophila genome revealed a number of a putative virulence factors,including a gene that heat-labile cytotonic enterotoxin (alt). Our study showed that all (16) isolates (100%) gave positive results to this gene,which gave molecular length (442)bp. Molecular detection of cytotonic enterotoxins gene (ast) was done for (16) A. hydrophila isolates and the results showed that all isolates have this gene (100%). The positive results for (ast) virulence were detected by the presence of (331) bp band compared with allelic ladder.

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.

Environmental factors on virulence of Aeromonas hydrophila

Aeromonas hydrophila are known for being opportunistic pathogens, harboring various virulence factors and triggering lesions and death in fish. The disease caused by bacteria can make fish inappropriate for human consumption, besides representing a risk to public health. The pathogenesis can be influenced by environmental variables, affecting fish productivity and mortality. The present study aimed to determine whether A. hydrophila harbor the virulence genes aerolysin, hydrolipase, elastase, lipase, cytotonic enterotoxin (ast), lateral flagellum (laf), and polar flagellum (fla) and to evaluate the influence of environmental variables on in vitro growth, in vivo virulence and expression of some of these genes. Polymerase chain reaction (PCR)-based screening for the presence of these virulence genes was performed on 35 isolates. Six isolates containing different profiles of virulence genes were tested for in vitro growth under different conditions of pH, temperature, and ammonia and for in vivo virulence under these same environmental conditions. RT-qPCR was used to quantify the expression of aerolysin, lipase, and fla genes. All the tested environmental factors influenced the growth of A. hydrophila, while pH and ammonia concentrations influenced the bacterial virulence. The expression of the fla gene increased when bacteria were grown in higher ammonia concentration. The mortality established by Aeromonas is influenced by several environmental factors pinpointing the importance of its control in fish farming to avoid higher economic loses associated to bacterial disease outbreaks.

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.