A Few Words about Aeromonas sp.
The members of the Aeromonas genus are water-borne, Gram-negative bacteria. They have been isolated from marine waters, rivers, lakes, ponds, swamps, sediments, chlorine water, water distribution systems, drinking water but not from deep sea; as well as they have been found in different types of food, animals, and various clinical samples. Aeromonas species are recognized as pathogenic to poikilothermic animals. In fish, they may cause furunculosis (nonmotile and psychrophilic species of A. salmonicida) as well as - motile aeromonas infection (MAI), chronic disease with skin ulceration, or motile aeromonad septicemia (MAS), acute systematic infection. In addition, the mesophilic strains are emerging as important pathogens in humans, causing a variety of extraintestinal and systemic infections. The most commonly described disease in human is the gastroenteritis; however, no adequate animal model is available to reproduce this illness caused by these microorganisms.
source A. Pękala
Acute systematic infection (MAS) in Carp
Aeromonas bacteria like other pathogens in aquaculture are opportunistic and may stay undetected until some stress make the fish susceptible to infection. Stress commonly include unsuitable temperature, pH, or salinity or quick shift within these parameters. Moreover, poor oxygenation; buildup of toxic chemicals, like ammonia; overcrovding; over or under feeding; excessive handling; and overall poor water quality had been described as possible agents of stress conditions.
The virulence of Aeromonas sp. is multifactorial. The main pathogenic factors associated with these bacteria are surface polysaccharides (capsule, lipopolysaccharide and glucan), protein S-layers, iron-binding systems, the III and VI secretion systems, fimbriae and other nonfilamentous adhesion factors. Moreover, it has been described that they produce, a wide range of exotoxins and extracellular enzymes including proteases, haemolysins.
LPS is the major component of the outer membrane of Gram-negative bacteria. Structurally it is composed of three parts: lipid A, a core oligosaccharide (OS), and O-antigen, which determines the specificity of each bacterial serotype. The O-chain structures, like any other polysaccharides, can be linear or branched and substituted by many different aglycones. The most common substituents are O- and N-acetyl, phosphate, and phosphoethanolamine groups.
Scheme of the Project
Bacterial strains received from National Veterinary Research Institute in Pulawy were isolated from fish culture in Poland
A study of heterogeneity of LPS of virulent and avirulent Aeromonas spp. using polyacrylamide gel
electrophoresis SDS-PAGE
Phenol/Water Extraction of LPS and Analysis of the chemical composition of LPS by gas chromatography-mass spectrometry GC-MC
Mild-acid degradation of the phenol-soluble LPS to obtain the O-specific polysaccharide fraction
Mass spectrometry (MALDI-TOF-MS, ESI-MS) and NMR spectroscopy analyses of the structure of the O-antigen
and the core region of LPS isolated from Aeromonas sp. strains belonging to the species A. sobria complex,
A. veronii bt. sobria and A. bestiarum
Detection and identification of antigenic determinants within the serogroups
important in the pathogenicity to fish using Western blotting with rabbit polyclonal antisera
Microscopic investigations of the co-culture between Aeromonas sp. bacteria and Acanthamoeba castellanii.
The presence of smooth, S-LPS and/or
protein S-layer correlates with an effectiveness of bacterial adhesion to amoeba cells
Aeromonas bacteria (red) inside amoeba vacuoles.
Detection and identification of immunochemical changes in LPS of Aeromonas bacteria released from amoeba cells by SDS-PAGE and immunoblotting analyses with polyclonal and monoclonal sera
RESULTS . . .
IMMUNOPROTECTION
Popularization aspect of the project results
Aeromonas sp. rods are ubiquitous in aquatic habitats and are a major cause of diseases in fish with great economic importance, e.g. carp and trout. Intensification of fish farming promotes the spread of infection and increasing environmental pollution with organic compounds facilitates pathogen growth. Recent research has indicated that the use of antibiotics and chemotherapeutics for elimination of pathogenic microorganisms in fisheries should be substantially limited. The application thereof leads to elimination of the infectious agent but only with a short-term effect. It weakens the natural defence system and increases fish susceptibility to secondary infections. Such practices contribute to an increase in environmental pollution as well.
Given the rapidly growing resistance of pathogenic bacteria to antibiotics and chemotherapeutics as well as the need for supplying healthy and unpolluted foods to the market, a decisive role in the prevention of bacterial fish diseases in fisheries should be played by prophylaxis and immunoprophylaxis that would increase the non-specific and/or specific immunity of fish.
The investigations focused on the use of Aeromonas sp. strains as a component of a polyvalent vaccine in immunoprophylaxis of ulcer diseases and haemorrhagic septicaemia in carp and furunculosis in trout require support from the scientific community. Failures in this field are associated with the large variety and variability of antigenic structures of e.g. the endotoxin of Aeromonas sp. bacteria, investigations of which are not easy. An effective vaccine should contain an immunogenic factor composed of strains with a representative antigenic profile characteristic of serogroups, among which pathogenic species are identified. This will result in achievement of protection against infections with already identified strains (homologous and heterologous) and new strain appearing in fisheries.
The structural analyses of LPS including chemical characterisation of Aeromonas sp. O-specific antigens and performed as part of the project will accelerate the research on the vaccine for immunoprophylaxis. The investigations also provide new knowledge and may contribute to elucidation of the molecular basis of relationship between Aeromonas rods and other Gram-negative bacteria.
The modern method for investigation of native Aeromonas endotoxin used in the project (MALDI-TOF mass spectrometry technique) facilitates diagnosis of the LPS antigen profile in strains involved in disease outbreaks. This more individualised approach can facilitate the monitoring of the breeding facilities to detect appearance of new pathogenic strains with altered antigenic properties. It also provides a starting point for preparation and development of effective autovaccines for individual fisheries. This practice seems to be a better solution for both economic and epidemiological reasons.
Another research aspect of the project is the importance the natural environmental co-existence of Aeromonas and Acanthamoeba for pathogenicity of the bacteria. In some Aeromonas strains, an effect of these interactions is transition from the metabolically active to inactive form that is impossible to culture in laboratory conditions. However, this form of the microorganism retains its virulence. Additionally, the completion of the intracellular bacterial cycle in the phagotrophic host cell may increase the pathogenicity of the bacteria. Then the amoebal cell plays a role of a safe niche for the growth of the microorganism and the vector transmitting it to the higher organism. Interactions between the partners depend on e.g. the surface structures of the bacteria, i.e. LPS and the S protein layer, which promote adhesion to the host cell. The aim of the study was to determine the importance of antigenic structures for the penetration, survival, and proliferation of Aeromonas rods in amoebal cells. Such investigations are important from the point of view of epidemiology and development of strategies for elimination of the pathogens and their vectors.
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The results obtained so far have been published in 4 original papers in renowned JCR journals in the field of immumochemical studies (Carbohydrate Research and Marine Drugs). The total IF of the papers is 10,278, and the MNiSW score is 130.
The unique structures of Aeromonas sp. O-specific antigens are available in the Bacterial Carbohydrate Structure DataBase, http://csdb.glycoscience.ru/.
The results of the experiments were presented at 7 conferences: 6 international and 1 national, in the form of 5 posters and 2 oral presentations as well as at scientific seminars in Poland and abroad. The measurable result of the project will include 5 Master theses and a habilitated doctor degree completed by the head of the project based on research achievements.
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Acknowledgement:
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This work was financially supported by the grant of National Science Centre (DEC-2011/03/B/NZ1/01203).
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