Hydrophila Isolates Research Articles

Antimicrobial resistance and the prevalence of integron in Aeromonas hydrophila from hemorrhagic diseased Pangasius catfish of the Mekong Delta

Antimicrobial resistance from bacterial pathogens is becoming a health concern in many countries. The purpose of this investigation was to assess antimicrobial resistance and find out the prevalence of class 1 integrons from A. hydrophila that led to hemorrhagic disease in intensively farmed Tra catfish in the Mekong Delta, Vietnam. The research showed that a total of 74 isolates were isolated and identified as A. hydrophila by the API 20E kit and using the amplification products of the aerolysin gene for confirmation. The susceptibility of all isolates was tested against fifteen antimicrobial agents using the disk diffusion method. Our study indicated that most strains were resistant to the following antibiotics: trimethoprimsulfamethoxazole, ampicillin, cefalexin, tetracycline, amoxicillin, chloramphenicol, florfenicol, neomycin, and gentamycin. In addition, the investigation also indicated that most A. hydrophila isolates displayed multiple antibiotic resistance phenotypes. The MAR (multiple antibiotic resistance) index was high, ranging from 0.40 to 0.66 for A. hydrophila isolates, which indicated that these isolates were exposed to high risk sources of contamination where antibiotics were commonly used. Among all isolates, 12.16% (9/74 isolates) of class 1 integrons were determined by polymerase chain reaction

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.

Epidemiological study of ticks harbouring Aeromonas hydrophila in areas endemic and non-endemic to Japanese-spotted fever.

Aeromonas spp. often cause life-threatening diseases, including necrotizing fasciitis, which may lead to septic shock and ultimately death. Aeromonas infections are believed to be transmitted via minor wounds or the consumption of fresh fish. However, after the detection of Aeromonas hydrophila in ticks in areas endemic to Japanese-spotted fever (JSF), a novel transmission route of A. hydrophila (i.e., via tick bites) has been proposed. We investigated the prevalence of A. hydrophila in ticks in areas endemic and not endemic to JSF in the Mie Prefecture, Japan. We collected ticks from endemic and nonendemic areas in summer and winter and assessed them for presence of A. hydrophila using polymerase chain reaction. Six A. hydrophila isolates were obtained from 95 ticks in endemic areas, whereas one A. hydrophila isolate was obtained from 142 ticks in non-endemic areas, in summer. All ticks that harboured A. hydrophila were Haemaphysalis longicornis (H.L); these ticks were almost at the larval stage and also carried Rickettsia spp. in the endemic area. In contrast, 51 and 41 ticks in the endemic and non-endemic areas were captured in winter, respectively; A. hydrophila was not detected in these. This study revealed the prevalence of tick-borne A. hydrophila. Therefore, the risk of transmission of A. hydrophila via a tick bite should be considered in the following conditions: areas abundant in H. L. harbouring Rickettsia spp., in areas endemic for JSF, presence of ticks in the larval stage and during the summer season.

Resistência de isolados de Aeromonas hydrophila a antimicrobianos e sanitizantes

ABSTRACT: In recent decades, Aeromonas hydrophila has emerged as a foodborne bacterial pathogen of public health concern, especially as it exhibits resistance to the major chemical sanitizers commonly used in the food industry. Meanwhile, this pathogen may be spread from diseased fish flesh through the contamination of equipment contact surfaces during food processing, thus posing a food safety risk. Thise determined the susceptibility profiles of retail fish-borne A. hydrophila isolates to 24 common antibiotics and five major sanitizers used in the food industry. The polymerase chain reaction technique was used to confirm all A. hydrophila isolates to the species level, and the agar diffusion method was applied to determine the antimicrobial susceptibility profiles. All isolates were confirmed to be A. hydrophila species. This bacterium was observed to have resistance to multiple antibiotics, with the highest resistance index being for those of the beta-lactam class. Additionally, the isolates showed high resistance to four of the five chemical sanitizers tested, with the highest resistance rate being toward sodium hypochlorite. The results suggested that A. hydrophila isolates with multiple resistance to the antimicrobials and main sanitizers used in the food industry can be found in retail fish sold in the Cuiabá region of Mato Grosso, Brazil.

Antibiotic resistance and virulence factor gene profile of A. hydrophila isolated from carp (Cyprinidae) suspected with hemorrhagic septicemia in Gilan, Iran.

The present study was conducted to determine the antibacterial resistance profile of Aeromonas hydrophila (n = 42) isolated from the 100 hemorrhagic septicemia-suspected carp in Gilan, Iran. The prevalence of class 1 and 2 integrons, antibiotic resistance genes (ARG) and virulence factor genes (VFG) among these isolates was investigated using PCR. Also, the possible association between the presence of VFGs and the antibiotic resistance profile of isolates was assessed. The majority of A. hydrophila isolates (83·33%) exhibited multi-drug resistance (MDR) profile, and all isolates were resistant to clindamycin, while all isolates were susceptible to amikacin. intI1 and intI2 gene was found in 26·2 and 4·8% isolates, respectively. This is the first report of the presence of the intI2 gene in A. hydrophila isolates in Iran. The blaTEM (40·5%) and tetA (33·3%) genes were found as the predominant ARGs. The most frequently detected VFGs were lip and ahh1(90·5%), while the examined isolates carrying at least three VFGs and the most prevalent VFGs profile was ast+, act+, alt+, ahhl+, aerA+, ahyB+ and lip+. The results of this study indicate a positive association between the presence of VFGs and antibiotic resistance, and most MDR A. hydrophila isolates showed high frequencies of VFGs.

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.

Clonal Dissemination of Aeromonas hydrophila With Binary Carriage of bla KPC-2-Bearing Plasmids in a Chinese Hospital.

Dissemination of the Klebsiella pneumoniae carbapenemase (KPC)-encoding gene among Enterobacterales is common but relatively rare in Aeromonas spp. In this study, we characterized two KPC-2-producing Aeromonas hydrophila strains (Ah2101 and Ah2111), each isolated from a patient in different intensive care units (ICUs) of a Chinese hospital. Whole-genome sequencing (WGS) revealed simultaneous carriage of the blaKPC−2 and imiH genes, both of which encode high-level carbapenem resistance in these two A. hydrophila isolates. The two isolates were shown to be clonally related and each isolate harbored two distinguishable blaKPC−2-bearing plasmids, only one of which was transferrable to A. hydrophila, but not Escherichia coli EC600 via conjugation. The genetic element that contains blaKPC−2 in these two plasmids, namely ISKpn27-ΔblaTEM−1-blaKPC−2-ISKpn6, was structurally identical, commonly detected in Enterobacterales, and associated with Tn3-based transposons. In addition, more than sixty putative genes that encode various virulence factors were identified in these two A. hydrophila isolates. This is the first study that reports clonal dissemination of carbapenem-resistant A. hydrophila strains carrying structurally different blaKPC−2-bearing plasmids. Further investigation is warranted to monitor the future transmission of blaKPC−2-bearing plasmids in A. hydrophila in clinical settings.

ANALYSIS OF THE PATHOGENESIS OF Aeromonas hydrophila IN THE AFRICAN CATFISH, Clarias gariepinus AND INVOLVEMENT OF THE TNF-a IN RESPONSE TO THE INFECTION

This research aimed to study the pathogenesis of Aeromonas hydrophila infection through two different routes of infection in African catfish and to find out the involvement of TNF-a in response to the infection. The experimental infection model was performed by clipping the caudal fin and immersing the fish in a medium with A. hydrophila and by intramuscular injection. Total plate count were used to investigate total and the distribution of A. hydrophila in the organs and TNF-a were observed using immune histochemistry. The results showed that the two types of infection were able to show typical A. hydrophila symptoms in experimental fish. Histological observation indicated that the two types of experimental infection resulted in systemic aeromoniasis infection. Total bacterial count results showed that A. hydrophila were detected three hours post-infection (hpi) in all organs, except for the kidney, in which detection started since hour 0, both in control and challenge fish. TNF-a were detected in all experimental fish and influenced by the number of bacteria, the function and tissue structure and of the organs. It can be concluded that artificial infection by clipping the caudal fin of Clarias gariepinus and immersing the fish in a medium with active A. hydrophila isolates cause systemic aeromoniasis infection in organs. Acute infection with Aeromonas hydrophila causes an increase in TNF-a production.

Examination of virulence and the effects of environmental conditions on the growth of Aeromonas hydrophila in farmed tilapia and histopathological changes in infected fish

The study aims to examine the pathogenicity, the effects of environmental conditions on the growth of Aeromonas hydrophila in tilapia, and histopathological changes in infected fish. A. hydrophila isolates, which were recovered from diseased tilapia samples collected at farming cages/ponds in several northern Vietnam provinces, were identified by biochemical tests, PCR confirmation, and sequencing of 16S rRNA and housekeeping genes (gyrB). Three representative strains after identification were subjected to evaluate the pathogenicity via challenge experiments using the intraperitoneal injection method and to determine the impacts of environmental factors on their growth. The present study showed an average LD50 value (lethal dose 50%) of A. hydrophila to tilapia at 4.6×105 CFU/fish, the infected fish in the challenge tests presented the clinical and gross lesions similar to the natural diseased fish, including haemorrhaging at the base of fins, skin, anal opening and in visceral organs, especially in liver and intestine. Histopathological examination of the diseased fish showed the hyperplasia of epithelial cells and haemorrhage of the gill filaments. The tissues of the liver, kidney, and spleen exhibited the lesion of haemorrhage, congestion, and degeneration, while the brain tissue appeared the colonisation by the bacteria. The A. hydrophila strains from tilapia showed a high tolerance to environmental conditions with the capacity to survive and multiply at a temperature of 15-45°C, a salinity of 0-60‰ and a pH of 5-10. The present results provide useful information to establish the strategies in prevention of A. hydrophila infection in tilapia and other freshwater fish.

Clinical and pathological investigations in ulcer disease of Cyrinus carpio caused by Aeromonas hydrophila

The Indian carps are the most important food fishes and they have a considerable economic impact on the country's economy. In several parts of the country, they are widely cultivated. The Indian Major Carps include the Catla catla, Labeo calbasu, Cirrhinus mrigala, and Labeo rohita, to name a few species. The fish Cyprinus carpio, both in the wild and in captivity, is vulnerable to Aeromonas hydrophila infections. Investigations carried out on Cyrinus carpio to better understand the implications of a pathogenic Aeromonas hydrophila isolates impacts on fish health revealed that the pathogen affects severely. The fish species that were injected with the bacterial suspension developed lesions of a similar nature, and dead fishes with ulcers on their bodies were found in all of the tanks. Even after 15 days of incubation and observation, no difference was observed in the fishes in the control group of fishes. Despite this, not all of the fish who got ulcers perished. Healing has been shown in some fish that had small ulcers, according to the research. Cyprinus carpio injected with a mixed bacterial suspension of AH-04 and AH-07 recorded a death rate was 65.0 percent.

Phenotypic characterization, genetic analysis and antibiotic sensitivity of Aeromonas hydrophila isolates causing dropsy in cultured Labeo rohita from Punjab, Pakistan

Motile Aeromonas septicemia (MAS) is a common freshwater fish disease and major threat to the aquaculture in Pakistan. The present study was carried out on suspected fish samples to isolate and characterize local strains of Aeromonas hydrophila, a key pathogen responsible for the said disease in aquacultured fishes. A total of ninety suspected fish specimens were collected from fish farms in Kasur, Okara and Gujranwala districts of Punjab, Pakistan from June 2018 to April 2019. The specimens were processed and A. hydrophila strains were isolated. The primary identification of sixty seven isolates were verified by colony morphology, microscopy and phenotypic characterization with ten biochemical reactions. The A. hydrophila strains of test samples were molecularly characterized by polymerase chain reaction (PCR) using 16S rRNA at desired size of 356bp. The PCR amplified product was subjected to DNA sequencing and phylogenetic analysis showed homology with related strains of Aeromonas spp. By antibiotic sensitivity test, the isolates were checked for nine antibiotics in which the pathogen was sensitive to four and resistant to five drugs. Results of genetic analysis confirmed strains as A. hydrophila which are useful to take preventive measures against the said disease.

Isolation, Characterization, and Metabolic Profiling of Ceratorhiza hydrophila from the Aquatic Plant Myriophyllum spicatum

The goal of the present study was to investigate the antibacterial properties, enzyme production, and metabolic profiling of a new Ceratorhiza hydrophila strain isolated from the submerged aquatic plant Myriophyllum spicatum. Furthermore, the fungus' morphological characterization and DNA sequencing have been described. The fungus has been identified and submitted to the GenBank as Ceratorhiza hydrophila isolate EG19 and the fungus ID is MK387081. The enzyme analyses showed its ability to produce protease and cellulase enzymes. According to the CSLI standard, the ethyl acetate extract of C. hydrophila showed intermediate antibacterial activity against Streptococcus pneumonia, Micrococcus luteus, and Staphylococcus aureus. Metabolic profiling has been carried out using 700 MHz NMR spectroscopy. Based on the 1H and 1H-13C heteronuclear single quantum coherence (HSQC) NMR data and NMR databases, 23 compounds have been identified. The identified metabolites include 31% amino acids, 9% sugars, 9% amines, 4% sugar alcohols, and 4% alkaloids. This is the first report for the metabolic characterization of C. hydrophila, which gave preliminary information about the fungus. It is expected that our findings not only will pave the way to other perspectives in enormous applications using C. hydrophila as a new promising source of antimicrobial agents and essential metabolites, but also it will be valuable in the classification and chemotaxonomy of the species.

Detection and distribution of virulence genes in Aeromonas hydrophila isolates causing infection in cultured carps.

Aeromonas hydrophila is a bacterium associated with many diseases and disorders such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. It bears several virulence factors including type III secretion system (T3SS), aerolysin, cytolytic enterotoxin and enzymes (e.g., hemolysins, lipase) that seem to play an important role in its pathogenesis. Detection of virulence markers by polymerase chain reaction (PCR) is a key procedure in defining the patho-genic ability of pathogenic bacteria and preparing a vaccine for its treatment. In this sense, this study was aimed to determine the frequency of virulence genes in isolates obtained from infected cultured carps in Khuzestan province. Out of 200 moribund carps with septicemic symptoms, 125 isolates were belonged to the motile aeromonads and 59 isolates were identified as A. hydrophila by biochemical methods. Finally, using PCR analysis, 31 isolates were identified as A. hydrophila. Five virulence genes were detected in these isolates including hemolysin, aerolysin, cytolytic enterotoxin and T3SS (aopB and ascV) by specific primers. Results showed that 23 (74.19%), 18 (58.06%), 16 (51.61%), 13 (41.63%) and 10 (32.25%) isolates possessed cytolytic enterotoxin, hemolysin, aerolysin, and T3SS genes, respectively. The results of the present study showed that among 31 isolates, only five isolates had all of dominant virulence genes. Thirteen other isolates had genotypes including hlyA+, aerA+, and act+. The remaining isolates had at least one virulence gene. This study showed that determination of the virulence genes by PCR can be a reliable method to identify a potential pathogenic Aeromonad strain.

Purification and structural characterization of lectin with antibacterial and anticancer properties from grubs of hide beetle, Dermestes frischii

Lectins or haemagglutinins are diverse classes of non-immune proteins; they bind to carbohydrates and are abundant in nature. In the present study, a coleopteran lectin from grubs of hide beetle, Dermestes frischii called DFL, was purified by glutaraldehyde (fixative-agent) fixed hen erythrocytes and characterized further for its functional properties. The purified DFL was stable between pH range 5 to 9 and heat-stable up to 50 °C. It was insensitive to EDTA and did not require any divalent cations. DFL native molecular mass was approximately 69 kDa with three different polypeptide subunits of 33 (pI ~4.4), 22 (pI ~6) and 14 (pI ~4.4) kDa. Haemagglutinating activity of DFL was highly inhibited by N-acetyl-D-glucosamine. DFL partial peptide sequences obtained from peptide mass fingerprinting experiments matched with amino acid sequences of lectins from different organisms confirmed its nature. Biological properties of purified DFL namely antibacterial and bacterial agglutination experiments revealed that DFL have both the effects against laboratory cultures of Aeromonas hydrophila, Enterococcus faecalis, Escherichia coli and habitat bacterial isolates of Staphylococcus cohnii and Bacillus cereus. In addition, the DFL exhibited substantial anticancer properties against HeLa cells. These results concluded that purified DFL could serve as a potent therapeutic agent for various biomedical applications.

Prediction and Analysis in silico of Genomic Islands in Aeromonas hydrophila.

Aeromonas are Gram-negative rods widely distributed in the environment. They can cause severe infections in fish related to financial losses in the fish industry, and are considered opportunistic pathogens of humans causing infections ranging from diarrhea to septicemia. The objective of this study was to determine in silico the contribution of genomic islands to A. hydrophila. The complete genomes of 17 A. hydrophila isolates, which were separated into two phylogenetic groups, were analyzed using a genomic island (GI) predictor. The number of predicted GIs and their characteristics varied among strains. Strains from group 1, which contains mainly fish pathogens, generally have a higher number of predicted GIs, and with larger size, than strains from group 2 constituted by strains recovered from distinct sources. Only a few predicted GIs were shared among them and contained mostly genes from the core genome. Features related to virulence, metabolism, and resistance were found in the predicted GIs, but strains varied in relation to their gene content. In strains from group 1, O Ag biosynthesis clusters OX1 and OX6 were identified, while strains from group 2 each had unique clusters. Metabolic pathways for myo-inositol, L-fucose, sialic acid, and a cluster encoding QueDEC, tgtA5, and proteins related to DNA metabolism were identified in strains of group 1, which share a high number of predicted GIs. No distinctive features of group 2 strains were identified in their predicted GIs, which are more diverse and possibly better represent GIs in this species. However, some strains have several resistance attributes encoded by their predicted GIs. Several predicted GIs encode hypothetical proteins and phage proteins whose functions have not been identified but may contribute to Aeromonas fitness. In summary, features with functions identified on predicted GIs may confer advantages to host colonization and competitiveness in the environment.

Genetic diversity and clonal relatedness of Aeromonas hydrophila strains isolated from hemorrhagic septicemia’s cases in common Carp (Cyprinus carpio) farms

The objective of this study was to determine molecular typing and comparison analysis of 24 Aeromonas hydrophila isolated from the diseased fish with hemorrhagic septicemia in freshwater ponds and cage in Mosul and Duhok cities, Iraq. A total of 24 A. hydrophila isolates that were collected from various fish ponds and cage, were used in this study. Identification of isolates was made by the standard microbiological and molecular methods. ERIC-PCR was done with different primers to establish the genetic relationship between strains. ERIC-PCR typing showed that 24 strains of A. hydrophila were classified into 11 ERIC types (genotypes). Genotypes 9 and 7 represented the most prevalent clone. All A. hydrophila strains that were isolated from the same fish were genetically diverse. There was minimal genetic similarity between some strains which were retrieved from the same geographical source area. Also, some isolates from different geographic source area were showed a 100% genetically similar. Aeromonas hydrophila was genotypically heterogeneous and clonally dispersed among different fish ponds and cage in Mosul and Duhok cities, Iraq. Besides, one fish can be infected with more than one strains of A. hydrophila.

Prevalence and Antibiogram of Vibrio parahaemolyticus and Aeromonas hydrophila in the Flesh of Nile Tilapia, with Special Reference to Their Virulence Genes Detected Using Multiplex PCR Technique.

Vibrio parahaemolyticus and Aeromonas hydrophila are major public health problems and the main cause of bacterial disease in Nile tilapia (Oreochromis niloticus). This study was conducted to determine the prevalence, antibiotic resistance and some virulence genes of both V. parahaemolyticus and A. hydrophila isolates from Nile tilapia. From Manzala Farm at Dakahlia governorate, 250 freshwater fish samples were collected. The confirmed bacterial isolates from the examined Nile tilapia samples in the study were 24.8% (62/250) for V. parahaemolyticus and 19.2% (48/250) for A. hydrophila. multiplex PCR, revealing that the tlh gene was found in 46.7% (29/62) of V. parahaemolyticus isolates, while the tdh and trh virulence genes were found in 17.2% (5/29). Meanwhile, 39.5% (19/48) of A. hydrophila isolates had the 16s rRNA gene and 10.5% (2/19) had the aerA and ahh1 virulence genes. The Multiple Antibiotic Resistance indices of V. parahaemolyticus and A. hydrophila were 0.587 and 0.586, respectively. In conclusion, alternative non-antibiotic control strategies for bacterial infections in farmed fish should be promoted to avoid multidrug-resistant bacteria. Therefore, it is suggested that farmers should be skilled in basic fish health control and that molecular detection methods are more rapid and cost-effective than bacteriological methods.

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.

Identification of novel vaccine candidates in the whole-cell Aeromonas hydrophila biofilm vaccine through reverse vaccinology approach

Biofilm vaccine has been recognised as one of the successful strategy to reduce the Aeromonas hydrophila infection in fish. But, the vaccine contains the protective and non-protective proteins, which may lead to show altered heterologous adaptive immunity response. Moreover, cross protection and effectiveness of previously developed biofilm vaccine was not tested against different geographical A. hydrophila isolates. Therefore, in the present study, whole-cell A. hydrophila biofilm vaccine was evaluated in rohu, vaccinated group showed increased antibody titer and protection against the different geographical A. hydrophila isolates namely KAH1 and AAH2 with 78.9% and 84.2% relative percentage survival, respectively. In addition, by using the immune sera of biofilm vaccinated group, a total of six protective proteins were detected using western blot assay. Further, the same proteins were identified by nano LC-MS/MS method, a total of fourteen candidate proteins showing the immunogenic property including highly expressed OMP's tolC, bamA, lamb, AH4AK4_2542, AHGSH82_029580 were identified as potential vaccine candidates. The STRING analysis revealed that, top candidate proteins identified may potentially interact with other intracellular proteins; involved in ribosomal and (tricarboxylic acid) TCA pathway. Importantly, all the selected vaccine candidate proteins contain the B-cell epitope region. Finally, the present study concludes that, whole-cell A. hydrophila biofilm vaccine able to protect the fish against the different geographical A. hydrophila isolates. Further, through reverse vaccinology approach, a total of fourteen proteins were identified as potential vaccine candidates against A. hydrophila pathogen.

Virulotyping and genetic diversity of Aeromonas hydrophila isolated from Nile tilapia (Oreochromis niloticus) in aquaculture farms in Egypt

This study focused on the genotyping of Aeromonas hydrophila isolated from diseased Nile tilapia, virulence genes, and their relation with motile aeromonad septicemia (MAS) clinical signs, and the resistance profiles of the recovered isolates to the frequently used antimicrobials in aquaculture. We determined the prevalence of A. hydrophila and the associated mortality in fish in various farms. We also investigated the extent of homogeneity of isolates sampled from various aquaculture farms and fish organs and how these variables would drive A. hydrophila heterogeneity to help developing control strategies against this infection. Of the 41 (41%) A. hydrophila isolates recovered by culture from diseased fish in four aquaculture fish farms (faced 35–50% mortality rates) located at different geographical regions in Egypt, 39 (39%) were confirmed by 16S rDNA-PCR amplification. Five distinct patterns were found for the distribution of virulence genes among 41% of A. hydrophila isolates. Thirteen isolates (33.33%) were positive for aer gene; 9 (23.1%) harbored lip gene, 2 (5.13%) carried hlyA gene, and one isolate (2.56%) was positive for ast gene. A positive correlation was found between hlyA and ast, and between lip and aer genes (r = 0.6 and 0.5, respectively). All isolates were resistant to ampicillin and amoxicillin. The least resistance was noted for doxycycline and gentamicin (7.7%) followed by streptomycin (10.3%), trimethoprim/sulfamethoxazole (17.9%), chloramphenicol (25.6%), colistin (43.6%), and erythromycin (47.7%). Most isolates were susceptible to ciprofloxacin. Multiple antimicrobial resistance index values ranged from 0·2–0·6 among multidrug-resistant (MDR) isolates (87·2%). Enterobacterial repetitive intergenic consensus (ERIC)-PCR allowed differentiating A. hydrophila isolates into 3 ERIC clusters, each contained isolates belonging to different farms and organs. The fingerprinting profiles of isolates were diverse without any obvious regional or organ-specific isolates. Our results indicated that sampling localities and sampled organs differ, albeit none substantially, in terms of the respective isolates heterogeneity. In conclusion, the high prevalence of A. hydrophila in Nile tilapia and its diverse antimicrobial resistance and virulence profile suggest the increased risk of this infection and raise concerns about its public health importance. The prudent use of antibiotics in the fish industry is now becoming mandatory to avoid the great losses in aquaculture due to MDR A. hydrophila. Our study also indicated the importance of ERIC genotyping for the analysis of A. hydrophila diversity during future molecular epidemiology investigations.