Virulence and antimicrobial resistance potential of Aeromonas spp. associated with shellfish.

Aeromonas spp. are associated with seafood-related outbreaks worldwide. In seafood industry, shellfish play a major role in global seafood production. With this emerging trend of shellfish consumption, shellfish-related bacterial infections are being reported frequently. Aeromonas spp. are natural contaminants found in shellfish. Although 36 species have been identified, some species including Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria have dragged major attention as foodborne pathogenic bacteria. The ability to elaborate a variety of virulence factors of Aeromonas spp. contributes to the pathogenic activities. Also, emerging antimicrobial resistance in Aeromonas spp. has become a huge challenge in seafood industry. Furthermore, multidrug resistance increases the risk of consumer health. Studies have supplied pieces of evidence about the emerging health risk of Aeromonas spp. isolated from seafood. Therefore, the present review was intended to highlight the prevalence, virulence and antimicrobial resistance of Aeromonas spp. isolated from various types of shellfish.

Characterization of virulence properties and multi-drug resistance profiles in motile Aeromonas spp. isolated from zebrafish (Danio rerio).

Aeromonas spp. are opportunistic pathogenic bacteria associated with a multitude of diseases in ornamental fish. In this study, virulence properties and antibiotic resistance patterns of 43 Aeromonas strains isolated from 46 zebrafish were investigated. The isolates were identified as Aeromonas veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), Aeromonas hydrophila (n = 8), A. caviae (n = 3), Aeromonas enteropelogenes (n = 2) and Aeromonas dhakensis (n = 1) by gyrB gene sequencing. The sequence divergence within and between the species ranged from 0-5·80% and 4·90-8·00%. Each species formed a distinct group in a neighbour-joining phylogenetic tree. The lipase production, biofilm formation, DNase activity, gelatinase production, caseinase production and ß-hemolysis were phenotypically observed in 34 (79·07%), 33 (74·74%), 30 (69·77%), 25 (58·14%), 22 (51·18%) and 21 (48·84%) isolates. The virulence genes were detected by polymerase chain reaction (PCR) in following frequencies- aer (86·05%), hlyA (83·72%), gcaT (83·72%), lip (72·09%), act (67·44%), fla (65·12%), ascV (58·14%), ast (55·81%), ser (41·86%), ahyB (39·53%) and alt (25·58%). Every isolate was resistant to at least four antibiotics in disk diffusion test. The multiple antibiotic resistance (MAR) index values ranged from 0·22-0·50 among the isolates. Our study suggests that zebrafish can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are Gram-negative and facultative anaerobic bacteria which are ubiquitous in aquatic environments. Virulence properties and antibiotic resistance of ornamental fish-borne Aeromonas spp. are poorly understood. The virulence factors as well as multiple antibiotic resistance profiles of zebrafish-borne Aeromonas spp. were characterized for the first time in Korea. Most of the isolates were positive for phenotypic virulence traits and harboured several virulence genes revealing the virulence potential of zebrafish-borne Aeromonas spp. Additionally, the high multiple antibiotic resistance (MAR) index values displayed by the isolates highlight the necessity of responsible use of antibiotics in the ornamental fish industry.

Aeromonas caviae alters the activities of ecto-enzymes that hydrolyze adenine nucleotides in fish thrombocytes.

It is recognized that the purinergic system, through the activities of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-nucleotidase), and ecto-adenosine deaminase (E-ADA), is involved in the regulation and modulation of the physiological and pathological events linked to hemostasis. This occurs due to the role of adenosine diphosphate (ADP) in the activation and recruitment of platelets, and the role of adenosine (Ado) in the inhibition of platelet activation. Thus, here we aimed to evaluate whether Aeromonas caviae infection impairs the ecto-enzymes of the purinergic system in fish thrombocytes and the involvement of this system in the hemorrhagic septicemia. The total number of fish thrombocytes decreased in infected animals compared to uninfected animals. Regarding the ecto-enzymes of the purinergic system, the E-NTPDase and E-5'-nucleotidase activities increased in infected animals compared to uninfected animals, while the E-ADA activity decreased. These findings show that adenine nucleotide hydrolysis is modified in the thrombocytes of fish experimentally infected with A. caviae, which impairs the coagulation process due the excessive hydrolysis of ADP, a molecule linked with activation and recruitment of thrombocytes at the site of vascular injury, and augmentation on Ado levels, a molecule linked with inhibitory effects on platelet activation and aggregation. In summary, the purinergic system might contribute to the occurrence of hemorrhagic frames in fish infected with A. caviae.

Xanthine oxidase activity affects pro-oxidative and pro-inflammatory profiles in spleen of silver catfish experimentally infected with Aeromonas caviae.

Several pieces of evidence have linked the involvement of xanthine oxidase (XO), a source of uric acid and reactive oxygen species (ROS), to pro-oxidative and pro-inflammatory effects observed during bacterial fish diseases. Thus, the aim of this study was to evaluate whether upregulation of splenic XO activity contributes to disease pathogenesis of Aeromonas caviae infection, as well as whether it may be considered a pathway involved in ROS and nitric oxide (NO) production. XO activity increased in the spleen of infected animals, as did the splenic levels of uric acid, ROS and metabolites of nitric oxide (NOx), compared to the uninfected control group. Based on this evidence, upregulation of splenic XO activity induces pro-oxidant and pro-inflammatory profiles in the spleen of fish infected by A. caviae due to excessive formation of uric acid. Moreover, excessive uric acid induces the release of pro-inflammatory mediators, such as ROS and NOx, which contribute to disease pathophysiology. In summary, upregulation of XO activity may be considered a pathway involved in ROS and NOx production.

Aeromonas caviae alters the cytosolic and mitochondrial creatine kinase activities in experimentally infected silver catfish: Impairment on renal bioenergetics.

Cytosolic and mitochondrial creatine kinases (CK), through the creatine kinase-phosphocreatine (CK/PCr) system, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. However, the effects of bacterial infections on the kidney remain poorly understood and are limited only to histopathological analyses. Thus, the aim of this study was to investigate the involvement of cytosolic and mitochondrial CK activities in renal energetic homeostasis in silver catfish experimentally infected with Aeromonas caviae. Cytosolic CK activity decreased in infected animals, while mitochondrial CK activity increased compared to uninfected animals. Moreover, the activity of the sodium-potassium pump (Na+, K+-ATPase) decreased in infected animals compared to uninfected animals. Based on this evidence, it can be concluded that the inhibition of cytosolic CK activity by A. caviae causes an impairment on renal energy homeostasis through the depletion of adenosine triphosphate (ATP) levels. This contributes to the inhibition of Na+, K+-ATPase activity, although the mitochondrial CK activity acted in an attempt to restore the cytosolic ATP levels through a feedback mechanism. In summary, A. caviae infection causes a severe energetic imbalance in infected silver catfish, which may contribute to disease pathogenesis.

Adhesion and cytotoxicity of Aeromonas caviae to rabbit intestinal epithelium ex vivo.

UNLABELLED: Aeromonads are considered potential pathogens for humans and animals and are responsible for the etiology of intestinal and extraintestinal diseases. The presence of Aeromonas spp. in food and water shows that it is an important vehicle of infection in humans. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The present study investigated the interaction of five Aeromonas caviae strains isolated from human diarrheic faeces with rabbit ileal and colonic mucosa ex vivo, using in vitro organ culture model. The in vitro adhesion assays using cultured tissue were performed with A. caviae strains co-incubated with intestinal fragments of ileum and colon over a period of 6 h. The fragments were analyzed by light and electron microscopy. All strains adhered to rabbit ileal and colonic mucosa ex vivo, with higher degree of adherence presented on colonic mucosa. The typical aggregative adherence pattern was observed among strains studied. Through electron and light microscopy, we observed extensive colonization of ileal and colonic mucosa, large mucus production, biofilm formation and morphological alterations such as intense vacuolization, structural disorganization, cell extrusion and destruction of the villi. These results demonstrate that in vitro organ culture of intestinal mucosa from rabbit may be used to investigate Aeromonas spp. PATHOGENESIS: Finally, our results support the pathogenic potential of Aeromonas emphasising their importance in public health.

Pan-genome analysis of Aeromonas hydrophila, Aeromonas veronii and Aeromonas caviae indicates phylogenomic diversity and greater pathogenic potential for Aeromonas hydrophila.

Aeromonas species are important pathogens of fishes and aquatic animals capable of infecting humans and other animals via food. Due to the paucity of pan-genomic studies on aeromonads, the present study was undertaken to analyse the pan-genome of three clinically important Aeromonas species (A. hydrophila, A. veronii, A. caviae). Results of pan-genome analysis revealed an open pan-genome for all three species with pan-genome sizes of 9181, 7214 and 6884 genes for A. hydrophila, A. veronii and A. caviae, respectively. Core-genome: pan-genome ratio (RCP) indicated greater genomic diversity for A. hydrophila and interestingly RCP emerged as an effective indicator to gauge genomic diversity which could possibly be extended to other organisms too. Phylogenomic network analysis highlighted the influence of homologous recombination and lateral gene transfer in the evolution of Aeromonas spp. Prediction of virulence factors indicated no significant difference among the three species though analysis of pathogenic potential and acquired antimicrobial resistance genes revealed greater hazards from A. hydrophila. In conclusion, the present study highlighted the usefulness of whole genome analyses to infer evolutionary cues for Aeromonas species which indicated considerable phylogenomic diversity for A. hydrophila and hitherto unknown genomic evidence for pathogenic potential of A. hydrophila compared to A. veronii and A. caviae.

DIARRHEA OUTBREAK IN PERNAMBUCO, BRAZIL, ASSOCIATED WITH A HEAT-STABLE CYTOTOXIC ENTEROTOXIN PRODUCED BY Aeromonas caviae.

In the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviae strains were examined. They originated from fecal specimens of patients with acute diarrhea during an outbreak in Brazil in 2004. Culture supernatants of fourteen strains (70%) caused fluid accumulation in rabbit ileal intestinal loops and in suckling mice assays, and also showed a cytotoxic activity in Vero and Caco-2 cells. The enterotoxic and cytotoxic factors were heat-stable after culture supernatants treatment at 100 ºC. The results revealed that A. caviae strains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil.

Diarrhea outbreak in Pernambuco, Brazil, associated with a heat-stable cytotoxic enterotoxin produced by Aeromonas caviae / O surto de diarreia em Pernambuco, Brasil é associado com uma enterotoxina citotóxica termo-estável produzida por Aeromonas caviae

SUMMARYIn the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviaestrains were examined. They originated from fecal specimens of patients with acute diarrhea during an outbreak in Brazil in 2004. Culture supernatants of fourteen strains (70%) caused fluid accumulation in rabbit ileal intestinal loops and in suckling mice assays, and also showed a cytotoxic activity in Vero and Caco-2 cells. The enterotoxic and cytotoxic factors were heat-stable after culture supernatants treatment at 100 ºC. The results revealed that A. caviaestrains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil.

RESUMOEm 2004 ocorreu um surto de diarreia aguda no Estado de Pernambuco, Brasil. Setenta por cento (14 dos 20) dos sobrenadantes de cultura de Aeromonas caviae,isoladas neste episódio induziram acúmulo de líquido em testes de alça ligada de intestino de coelhos, assim como em teste em camundongos recém-nascidos. Os mesmos sobrenadantes mostraram também atividade citotóxica em células de Vero e Caco-2, mas não em células HeLa e HEp2. As atividades enterotóxicas e citotóxicas mantiveram-se mesmo após o aquecimento a 100 ºC dos sobrenadantes de cultura. Este trabalho revela a expressão de um provável fator diarreiogênico: uma enterotoxina-citotóxica termo-estável, produzida por A. caviaeque pode ser associada ao surto de diarreia ocorrido no Brasil. Atualmente estamos purificando esta enterotoxina termo-estável, com o objetivo de elucidar seu papel como fator de virulência na diarreia causada por A. caviae.

Different clinical characteristics among Aeromonas hydrophila, Aeromonas veronii biovar sobria and Aeromonas caviae monomicrobial bacteremia.

This study aimed to compare the clinical presentations of Aeromonas hydrophila, A. veronii biovar sobria and A. caviae monomicrobial bacteremia by a retrospective method at three hospitals in Taiwan during an 8-yr period. There were 87 patients with A. hydrophila bacteremia, 45 with A. veronii biovar sobria bacteremia and 22 with A. caviae bacteremia. Compared with A. hydrophila and A. veronii biovar sobria bacteremia, A. caviae bacteremia was more healthcare-associated (45 vs 30 and 16%; P = 0.031). The patients with A. caviae bacteremias were less likely to have liver cirrhosis (27 vs 62 and 64%; P = 0.007) and severe complications such as shock (9 vs 40 and 47%; P = 0.009) and thrombocytopenia (45 vs 67 and 87%; P = 0.002). The APACHE II score was the most important risk factor of Aeromonas bacteremia-associated mortalities. The APACHE II scores of A. caviae bacteremias were lower than A. hydrophila bacteremia and A. veronii biovar sobria bacteremia (7 vs 14 and 16 points; P = 0.002). In conclusion, the clinical presentation of A. caviae bacteremia was much different from A. hydrophila and A. veronii biovar sobria bacteremia. The severity and mortality of A. caviae bacteremia were lower than A. hydrophila or A. veronii biovar sobria bacteremia.

Association of Aeromonas caviae polar and lateral flagella with biofilm formation.

AIM: To evaluate the association of the polar and lateral flagella with biofilm formation on plastic surfaces in 76 Aeromonas caviae strains isolated from environment (lagoon water), food (vegetables, fish and cheese) and human source (faeces). METHODS AND RESULTS: Both polar (flaA) and lateral (lafA) flagellin genes have been investigated by means of PCR and colony blot hybridization assays. The ability to form biofilm in polystyrene microtitre plates was evaluated and correlated with the presence and absence from these genes. The flaA and lafA genes had a frequency of 94% and 71%, respectively. All lafA(+) strains were also flaA(+) . Biofilm formation was observed in 72% of strains. Ninety-four per cent of flaA(+) lafA(+) strains could form biofilm and those that presented an intense biofilm production harboured both genes. All flaA(-) lafA(-) isolates, as well as 76% of flaA(+) lafA(-) strains, were incapable of forming biofilm. All the fish strains were flaA(+) lafA(+) and displayed higher biofilm formation (88%). Lagoon water samples exhibited lower positivity rate for the lafA gene (57%) and decreased ability to produce biofilm (39%). CONCLUSIONS: Both polar and lateral flagellar function contribute to biofilm formation in Aer. caviae strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence for the association of both flagella with biofilm formation, a factor required for pathogenicity of Aer. caviae strains of varied sources, especially food and human.