The lipopolysaccharide of Aeromonas spp: structure-activity relationships

Marine microorganisms, including Aeromonas, are a source of compds. for drug development that have generated great expectations in the last decades. Aeromonas infections produce septicemia, and ulcerative and haemorrhagic diseases in fish. Among the pathogenic factors assocd. with Aeromonas, the lipopolysaccharides (LPS)​, a surface glyconconjugate unique to Gram-​neg. bacteria consisting of lipid A (lipid anchor of the mol.)​, core oligosaccharide and O-​specific polysaccharide (O antigen)​, are key elicitors of innate immune responses. The chem. structure of these three parts has been characterized in Aeromonas. Based on the high variability of repeated units of O-​polysaccharides, a total of 97 O-​serogroups have been described in Aeromonas species, of which four of them (O:11; O:16; O:18 and O:34) account for more than 60​% of the septicemia cases. The core of LPS is subdivided into two regions, the inner (highly conserved) and the outer core. The inner core of Aeromonas LPS is characterized by the presence of 3-​deoxy-​d-​manno-​oct-​2-​ulosonic (ketodeoxyoctonic) acid (Kdo) and l-​glycero-​d-​manno-​Heptoses (l,​d-​Hep)​, which are linked to the outer core, characterized by the presence of Glc, GlcN, Gal, and GalNAc (in Aeromonas salmonicida)​, d,​d-​Hep (in Aeromonas salmonicida)​, and l,​d-​Hep (in Aeromonas hydrophila)​. The biol. relevance of these differences in the distal part of the outer core among these species has not been fully assessed to date. The inner core is attached to the lipid A, a highly conserved structure that confers endotoxic properties to the LPS when the mol. is released in blood from lysed bacteria, thus inducing a major systemic inflammatory response known as septic or endotoxic shock. In Aeromonas salmonicida subsp. salmonicida the Lipid A components contain three major lipid A mols., differing in acylation patterns corresponding to tetra-​, penta- and hexa-​acylated lipid A species and comprising of 4'-​monophosphorylated β-​2-​amino-​2-​deoxy-​d-​glucopyranose-​(1→6)​-​2-​amino-​2-​deoxy-​d-​glucopyranose disaccharide. In the present review, we discuss the structure-​activity relationships of Aeromonas LPS, focusing on its role in bacterial pathogenesis and its possible applications.

Enteropatógenos bacterianos em peixes criados em uma estação de reciclagem de nutrientes e no ecossistema relacionado

Avaliou-se a presença de enteropatógenos bacterianos em 72 amostras obtidas a partir de peixes criados em sistema de reciclagem de nutrientes, em estação experimental, no município de Petrópolis, RJ. Paralelamente, foram obtidas amostras do lodo utilizado como adubo orgânico e da cama de aves localizada na área interna dos tanques criatórios. A metodologia empregada incluiu o pré-enriquecimento em Caldo Lactosado e Água Peptonada Tamponada, seguido de enriquecimento em Água Peptonada Alcalina (pH 8,4-8,6) e subseqüente semeadura em Agar GSP para o isolamento de Aeromonas spp. e Plesiomonas shigelloides. Para os demais microrganismos, alíquotas de 1ml foram inoculadas nos meios de enriquecimento Caldo Rappaport-Vassiliadis e Caldo Tetrationato de Kauffmann com posterior semeadura em Agar Entérico Hektoen e Agar Salmonella-Shigella. Com a finalidade de monitorar o índice de coliformes fecais, visando conhecer a qualidade da água para este sistema, paralelamente à coleta de peixes foram avaliadas amostras de água dos tanques criatórios e de macrófitas. No cômputo geral foram isoladas 116 cepas de enteropatógenos bacterianos, destacando-se Aeromonas spp (67,2%) com 9 espécies (A. veronii biogrupo sobria, A. hydrophila, A. sobria, A. trota, A.eucrenophila, A. veronii biog. veronii, A. media, A. caviae e A jandaei) e Aeromonas spp., seguido de Edwardsiella tarda (16,4%), Plesiomonas shigelloides (12,9%) e Salmonella (3,4%). A análise da qualidade da água empregada no sistema revelou, de um modo geral, índices mais elevados de coliformes fecais nos tanques dos peixes (>1800/100 ml).

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

Multiple factors can be involved in the virulence processes of Aeromonas hydrophila. The objective of the present paper was to verify the presence of aerolysin, hidrolipase, elastase and lipase virulence genes through the polymerase chain reaction (PCR) in A. hydrophila isolates obtained from fish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia fingerlings. One hundred and fourteen isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using specific primers described in the literature. For in vivo tests Nile tilapia fingerlings were used. From the PCR tests, negative isolates for all genes tested were selected, positive isolates for two genes (aerolysin and elastase) and positive for the four genes tested. These were inoculated at a concentration of 10(8) UFC/ml into the tilapias, considered as treatments; another group of animals was used as control (with inoculation of saline solution). In all, 12 distinct standards regarding the presence of virulence factors in isolates from A. hydrophila, were observed. Of the 114 isolates analyzed, 100 (87.72%) presented at least one of the virulence factors under study. The virulence factors were widely distributed among the A. hydrophila isolates. Aerolysin was the most frequent virulence factor present in the isolates analyzed. A. hydrophila led to the mortality of the Nile tilapia fingerlings, regardless of the absence or quantity of virulence genes tested.

Ascophyllum nodosum in the diet of tilapia (Oreochromis niloticus) and its effect after inoculation of Aeromonas hydrophila

The objective of this study was to evaluate the effect of Ascophyllum nodosum brown seaweed meal (FAM) on the health of Nile tilapia submitted to inoculation with Aeromonas hydrophila. The experiment was conducted for a period of 40 days using 120 Nile tilapia fingerlings, with age of 40 days, distributed in 20 tanks. A diet including Ascophyllum nodosum seaweed meal at 20g.kg-1 and a control diet (without FAM) were provided which constituted the treatments. Thirty days after beginning the experiment, A. hydrophila was inoculated by bacterial inoculum diluted in sterile saline solution at a concentration of 10(6) CFU ml-1. Except for the width, which was greater for the treatment with the provision of FAM (P<0.05), there was no influence on the performance parameters of the fingerlings, but the occurrence of lesions in animals inoculated with A. hydrophila and fed with FAM was lower and they also exhibited a decline in the lesions in a shorter period of time than the control group. FAM prevents hepatopancreatic congestion in infected animals. Ascophyllum nodosum brown seaweed meal reduced the number of lesions in fish in a shorter time when compared to the control group.

Formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo

O objetivo desta pesquisa consistiu em avaliar a formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo. As variáveis em estudo consistem no cultivo monoespécie e combinado, dos referidos microrganismos e nas temperaturas de 4, 7 e 18 °C. Recipientes contendo 1000 mL de leite, densidade populacional de 10(5) UFC.mL-1 de cada microrganismo e 10 cupons de aço inoxidável (10 × 20 mm) foram lacrados e armazenados, sob agitação de 60 rpm, por um período de 10 dias. As análises ocorreram a cada 48 horas. Células sésseis de A.hydrophila e S. aureus foram enumeradas através do plaqueamento seletivo em ágar m-Aeromonas selective e Baird-Parker, respectivamente. Estudos sobre o tempo de geração, enumeração de células planctônicas e observação dos cupons através da microscopia eletrônica de varredura foram conduzidos. S. aureus, em monocultivo, formou biofilme a 18 °C e a 7 °C. Para 4 °C, foi observado um processo de adesão. A presença de A. hydrophila reduziu o desempenho de S. aureus. Nesta condição de cultivo multiespécie houve formação de biofilme a 18 °C. A. hydrophila, tanto em monocultivo quanto em presença de S. aureus, formou biofilme em todas as condições pesquisadas.

Characterization of antimicrobial resistance in Aeromonas and Vibrio isolated in Canada from fish and seafood

Plusieurs études ont examiné la sensibilité aux antimicrobiens chez les bactéries d’organismes provenant de produits issus de l’aquaculture ou de leur environnement. Aucune information n’est cependant disponible concernant la résistance aux antimicrobiens dans les bactéries de la flore de poissons ou de fruits de mer vendus au détail au Canada. C’est particulièrement vrai en ce qui a trait aux bactéries des genres Aeromonas et Vibrio, dont certaines espèces sont des agents pathogènes zoonotiques connus. Au cours de cette étude, la sensibilité aux antimicrobiens d’isolats d’Aeromonas spp. et de Vibrio spp. provenant de poissons et de crevettes domestiques et importés a été mesurée à l’aide de techniques de micro dilution en bouillon et/ou de diffusion sur disque. Les classes d’antimicrobiens examinés comprenaient les tétracyclines (TET), les inhibiteurs de la voie des folates (sulfadiméthoxine-triméthoprime, SXT), le florfenicol (FLO), et les quinolones (acide nalidixique / enrofloxacine, NA/ENO). Des valeurs seuils épidémiologiques pour Aeromonas et Vibrio ont été établies en utilisant la méthode d’interprétation normalisée des données de résistance provenant de diffusion sur disque. La recherche de gènes de résistance associés au profil de résistance des isolats a été effectuée en utilisant des PCRs et des puces ADN. Le nombre d’isolats résistants aux divers antimicrobiens parmi les 201 isolats d’Aeromonas et les 185 isolats de Vibrio étaient respectivement les suivants: TET (n=24 et 10), FLO (n=1 et 0), SXT (n=2 et 8), NA (n=7 et 5) et ENO (n= 5 et 0). Diverses associations de gènes tet(A), tet(B), tet(E), floR, sul1, sul2, et intI1 ont été détectées, les gènes tet(E), intI1, sul2 et tet(B) étant les plus communs. Les espèces d’Aeromonas et de Vibrio isolées de poissons au détail et de fruits de mer peuvent héberger une variété de gènes de résistance, bien que peu fréquemment. Le risque que représente ces gènes de résistance reste à évaluer en considérant le potentiel infectieux des bactéries, l’utilisation des ces agents antimicrobiens pour le traitement des maladies en aquaculture et en médecine humaine et leur rôle en tant que réservoir de la résistance antimicrobienne.

Distribution, Extracellular Virulence Factors and Drug Resistance of Motile Aeromonads in Fresh Water Ornamental Fishes and Associated Carriage Water

During last decades there has been a continuous growth of aquaculture industries all over the world and taking into consideration the spurt in freshwater ornamental fish aquaculture and trade in Kerala, the present study was aimed to assess the prevalence of various motile Aeromonas spp. in fresh water ornamental fishes and associated carriage water. The extracellular virulence factors and the antibiogram of the isolates were also elucidated. Various species of motile aeromonads such as Aeromonas caviae, A. hydrophila, A. jandaei, A. schubertii, A. sobria, A. trota and A. veronii were detected. Aeromonas sobria predominated both fish and water samples. Extracellular enzymes and toxins produced by motile aeromonds are important elements of bacterial virulence. The production of extracellular virulence factors - proteases, lipase, DNase and haemolysin by the isolates were studied. All the isolates from both fish and water samples produced gelatinase and nuclease but the ability to produce lipase, caseinase and haemolysins was found to vary among isolates from different sources. Among the 15 antibiotics to which the isolates were tested, all the isolates were found to be sensitive to chloramphenicol, ciprofloxacin and gentamicin and resistant to amoxycillin. Local aquarists maintain the fish in crowded stressful conditions, which could trigger infections by the obligate/ opportunistic pathogenic members among motile aeromonads

Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish and prawn of South India

A total of 319 strains of Aeromonas hydrophila were isolated from 536 fish and 278 prawns for a 2-year period. All the strains were tested for resistance to 15 antibiotics and 100% of the strains was resistant to methicillin and rifampicin followed by bacitracin and novobiocin (99%). Only 3% of the strains exhibited resistance against chloramphenicol. The multiple antibiotic resistance (MAR) indexing of A. hydrophila strains showed that all of them originated from high-risk sources

Prevalence of Aeromonas hydrophila in fish and prawns from the seafood market of Coimbatore, South India

Five hundred and thirty six samples offishes and 278 prawn samples from the major fish market ofCoimbatore, South India, were analysed for the prevalence of Aeromonas hydrophila over a period of2 years (June 1997–May 1999). The prevalence level of A. hydrophila varied from 17.62% in prawns to 33.58% in fishes. More than 30% of the popular table fishes such as Sardinella longiceps, Rastrelliger kanagurta, Mugil cephalus and Caranx sexfasciatus were tested positive for this organism. Among the different species of the prawns analysed, Penaeus semisulcatus showed higher incidence (23.52%). Seasonal variation in the prevalence levels of A. hydrophila in fish and prawns revealed a higher prevalence during the monsoon season during 1997–98 and 1998–99. Of the different body parts of the fishes analysed for A. hydrophila, the intestinal samples showed higher prevalence (38.43%), followed by body surface (32.46%) and gill (29.10%). Considering the psychrotrophic nature and role of A. hydrophila as a pathogen ofemerging importance, the considerably high levels ofthis organism in a popular food item such as fish and prawn raises serious concern

Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD50 values of 104.07–105.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.

Marine yeast Candida MCCF 101 as feed supplement in aquaculture: nutritional quality, optimization of large scale production and evaluation of its protective effect on Koi carp from Aeromonas infection

Marine yeast have been regarded as safe and showing a beneficial impact on biotechnological process. It provides better nutritional and dietary values indicating their potential application as feed supplements in aquaculture. Brown et al. (1996) evaluated all the marine yeasts characterised with high protein content, carbohydrate, good amino acid composition and high levels of saturated fats. However, there is paucity of information on marine yeasts as feed supplements and no feed formulation has been found either in literature or in market supplemented with them. This statement supported by Zhenming et al. (2006) reported still a lack of feed composed of single cell protein (SCP) from marine yeasts with high content of protein and other nutrients. Recent research has shown that marine yeasts also have highly potential uses in food, feed, medical and biofuel industries as well as marine biotechnology (Chi et al., 2009; 2010). Sajeevan et al. (2006; 2009a) and Sarlin and Philip (2011) demonstrates that the marine yeasts Candida sake served as a high quality, inexpensive nutrient source and it had proven immunostimulatory properties for cultured shrimps. This strain has been made part of the culture collection of National Centre for Aquatic Animal Health, Cochin University of Science and Technology as Candida MCCF 101. Over the years marine yeasts have been gaining increased attention in animal feed industry due to their nutritional value and immune boosting property.Therefore, the present study was undertaken, and focused on the nutritional quality, optimization of large scale production and evaluation of its protective effect on Koi carp from Aeromonas infection

Distribution, extracellular virulence factors and antibiogram of motile aeromonads in fresh water ornamental fishes and immune response of Cyprinus carpio against Aeromonas hydrophila infection

The thesis deals with the prevalence and distribution of motile aeromonads in selected ornamental fishes. The presence of motile aeromonads in ornamental fishes and associated carriage water is well documented. Though aeromonads are a part of autochthonous flora of natural waters, disease outbreak occurs as a result of environmental stress on the cultured species and virulence of the pathogens. While ornamental aquaculture in many parts of the world is highly organized and practiced scientifically, it is highly unorganized in India. The culture ponds/tanks are often maintained in very poor manner and the fishes are subjected to high degree of stress during transportation from the production facility to retail vendors. The situation is no better at retail outlets, where fishes are maintained in crowded condition without proper aeration or food. All these could result in high prevalence of diseases caused by motile aeromonads. No systematic study has been carried out to understand the prevalence of motile aeromonads in ornamental fishes and carriage water . It also gives an account of the production of extracellular virulence factors and the antibiogram of the different species of motile aeromonads isolated. The growth characteristics and virulence potential of a representative strain of Aeromonas hydrophila is also studied. The nucleotide sequencing of the strain was carried out and sequences deposited in Genbank. Survival and immune response of Cyprinus carpio under different stress conditions and on probiotic treatment with Bacillus NL110, when challenged with A. hydrophila is also dealt within this thesis.

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in Sao Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A. allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviare, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.

Dynamics of Aeromonas species isolated from wastewater treatment system

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of Sao Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism`s dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hly A/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hly A/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.