Aeromonas flagella and colonisation mechanisms.

Aeromonas species are inhabitants of aquatic environments and are able to cause disease in humans and fish among other animals. In aquaculture, they are responsible for the economically important diseases of furunculosis and motile Aeromonas septicaemia (MAS). Whereas gastroenteritis and wound infections are the major human diseases associated with the genus. As they inhabit and survive in diverse environments, aeromonads possess a wide range of colonisation factors. The motile species are able to swim in liquid environments through the action of a single polar flagellum, the flagellin subunits of which are glycosylated; although essential for function the biological role of glycan addition is yet to be determined. Approximately 60% of aeromonads possess a second lateral flagella system that is expressed in viscous environments for swarming over surfaces; both flagellar systems have been shown to be important in the initial colonisation of surfaces. Subsequently, other non-flagellar colonisation factors are employed; these can be both filamentous and non-filamentous. The aeromonads possess a number of fimbrial systems with the bundle-forming MSHA type IV pilus system, having a major role in human cell adherence. Furthermore, a series of outer-membrane proteins have also been implicated in the aeromonad adhesion process. A number of strains are also capable of cell invasion and that maybe linked with the more invasive diseases of bacteraemia or wound infections. These strains employ cell surface factors that allow the colonisation of these niches that protect them from the host's immune system such as S-layers, capsules or particular lipopolysaccharides.

Aeromonas species associated with necrotizing enteritis and septicemia in an adult male ostrich (Struthio camelus).

A deceased 10-yr-old male ostrich was diagnosed with severe necrotizing enteritis and septicemia. The bird was inappetent for 3 wk and had neurologic signs 2 days prior to death. Macroscopically, no significant lesions were noted aside from congestion of the liver, kidneys, and spleen. Histopathology revealed severe fibrinonecrotic enteritis,associated with large numbers of gram-negative bacteria, multifocal fibrinoid necrosis in portal arteries, accumulation of fibrin in hepatic sinusoids, myocardial degeneration, and necrosis. There was also squamous metaplasia in the glands of the esophagus and external ears. A gram-negative rod was isolated in pure culture from intestine, liver, lungs, and trachea and identified as an Aeromonas species. The concentration of vitamin A in the liver was extremely low. The lesions seen in the intestine and liver and the isolation of an Aeromonas sp. from various tissues strongly suggest that this bacterium was the cause of the necrotizing enteritis, septicemia, and death of this ostrich. Vitamin A deficiency might have predisposed the bird to the Aeromonas infection.

Aeromonas piscicola sp. nov., isolated from diseased fish.

Four Aeromonas strains (S1.2(T), EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA-DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2(T) (=CECT 7443(T), =LMG 24783(T)) as the type strain.

Characterization of electrochemical activity of a strain ISO2-3 phylogenetically related to Aeromonas sp. isolated from a glucose-fed microbial fuel cell.

The microbial communities associated with electrodes in closed and open circuit microbial fuel cells (MFCs) fed with glucose were analyzed by 16S rRNA approach and compared. The comparison revealed that bacteria affiliated with the Aeromonas sp. within the Gammaproteobacteria constituted the major population in the closed circuit MFC (harvesting electricity) and considered to play important roles in current generation. We, therefore, attempted to isolate the dominant bacteria from the anode biofilm, successfully isolated a Fe (III)-reducing bacterium phylogenetically related to Aeromonas sp. and designated as strain ISO2-3. The isolated strain ISO2-3 could grow and concomitantly produce current (max. 0.24 A/m(2)) via oxidation of glucose or hydrogen with an electrode serving as the sole electron acceptor. The strain could ferment glucose, but generate less electrical current. Cyclic voltammetry supported the strain ISO2-3 was electrically active and likely to transfer electrons to the electrode though membrane-associated compounds (most likely c-type cytochrome). This mechanism requires intimate contact with the anode surface. Scanning electron microscopy revealed that the strain ISO2-3 developed multiplayer biofilms on the anode surface and also produced anchor-like filamentous appendages (most likely pili) that may promote long-range electron transport across the thick biofilm.

Accumulation of silver(I) ion and diamine silver complex by Aeromonas SH10 biomass.

The biomass of Aeromonas SH10 was proven to strongly absorb Ag+ and [Ag(NH3)2]+. The maximum uptake of [Ag(NH3)2]+ was 0.23 g(Ag) g(-1)(cell dry weight), higher than that of Ag+. Fourier transform infrared spectroscopy spectra analysis indicated that some organic groups, such as amide and ionized carboxyl in the cell wall, played an important role in the process of biosorption. After SH10 cells were suspended in the aqueous solution of [Ag(NH3)2]+ under 60 degrees C for more than 12 h, [Ag(NH3)2]+ was reduced to Ag(0), which was demonstrated by the characteristic absorbance peak of elemental silver nanoparticle in UV-VIS spectrum. Scanning electron microscopy and transmission electron microscopy observation showed that nanoparticles were formed on the cell wall after reduction. These particles were then confirmed to be elemental silver crystal by energy dispersive X-ray spectroscopy, X-ray diffraction, and UV-VIS analysis. This study demonstrated the potential use of Aeromonas SH10 in silver-containing wastewater treatment due to its high silver biosorption ability, and the potential application of bioreduction of [Ag(NH3)2]+ in nanoparticle preparation technology.

Aplicação de métodos moleculares no estudo de organismos do gênero Aeromonas / Application of molecular methods in the study of organisms of the Aeromonas sort

As doenças emergentes são o foco de crescente atenção entre os profissionais de saúde pública.A emergência de novas doenças , ressurgimento de velhas, e as já reconhecidas, representam desafios para o entendimento da cadeia ecológica casual, que incluem componentes sociais, econômicos, ambientais e biológicos.Aeromonas estão amplamente distribuídas no ambiente aquático.Algumas espécies estão associadas com uma ampla variedade de doenças em animais de sangue frio ou quente.Em humanos esses organismos são responsáveis por gastrenterites e infecções extrintestinais incluindo septicemia.A taxonomia do gênero e a identificação de algumas cepas são consideradas tarefas difíceis devido às reações atípicas observadas para esses organismos.A limitação associada aos métodos convencionais tem estimulado o interesse na aplicação de métodos moleculares para estabelecer o posicionamento taxonômico de microrganismos e também na determinação de relação, em estudos epidemiológicos.Essas técnicas vêm, crescentemente sendo consideradas no estudo de cepas de Aeromonas.Nesse estudo o sequenciamento do fragmento 16S rDNA e a hibridização DNA/DNA foram aplicados para Aeromonas clínicas e ambientais para a confirmação do posicionamento taxonômico das cepas.Eletroforese em campo pulsado (PFGE)e PCR com base em sequências repetitivas do genoma ERIC, BOX, e REP foram utilizados para gerar um padrão de bandas e estabelecer a relação entre as cepas.O posicionamento taxonômico de cepas de Aeromonas atípicas, isoladas de amostras diarréicas e ambientais, foi confirmado pelo sequenciamento e hibridização DNA/DNA.Uma nova espécie de Aeromonas foi obtida das amostras de fezes diarréicas e, a presença de A. veronii sobria e A. media no Continente Antártico foram confirmadas.O perfil de bandas (fingerprinting) demonstrou sua utilidade em estudos epidemiológicos do gênero Aeromonas e a relação genética muito próxima entre os isolados das amostras fecais sugerindo que os casos sejam considerados...

Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp.

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.

Lateral flagella and swarming motility in Aeromonas species.

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.

Virulence factors-pathogenicity relationships for Aeromonas species from clinical and food isolates.

The presence of virulence factors in 96 Aeromonas strains isolated from food and clinical samples was studied. Neither cytotoxic activity and hydrophobicity, not the presence of pili or an extra surface layer made it possible to establish differences between food and clinical strains. Statistical studies showed that cytotoxin production was associated with a positive Voges-Proskauer reaction, inability to ferment arabinose and a positive lysine decarboxylation. Therefore, when comparing cytotoxic clinical and food strains with lysine decarboxylation phenotype, there was a significant difference (p < 0.05) between the two groups. The association of a cytotoxin production and lysine decarboxylation character should thus be considered as a possible virulence marker.

Adherence of Aeromonas caviae to human cell lines Hep-2 and Caco-2.

Adherence of Aeromonas caviae to HEp-2 and Caco-2 cell monolayers was investigated with 24 clinical isolates. Growth phase, temperature, multiplicity of infection and length of incubation affected adherence. Treatment of the bacteria with trypsin, sodium metaperiodate, mechanical shearing and the addition of cytochalasin B and cycloheximide to the monolayer significantly reduced the adherence capabilities of the strains investigated. The use of chloramphenicol to inhibit protein synthesis reduced the adhesive capabilities of bacteria grown in liquid medium and those subjected to mechanical shearing. Light microscopy, scanning and transmission electron microscopy were employed in the investigation of bacteria-bacteria and bacteria-monolayer interactions and indicated similarities with the aggregative adherence patterns of the Enterobacteriaceae. The presence of extracellular bacterial appendages and their correlation with increased adhesive capacity may indicate a role in the process of adherence.

Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface.

Physical and functional S-layer reconstitution in Aeromonas salmonicida.

The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.

Molecular characterization of an Aeromonas salmonicida mutant with altered surface morphology and increased systemic virulence.

The asoA gene of Aeromonas salmonicida is located approximately 7 kb downstream of the A-layer structural gene, vapA. A 6 kb BamHI fragment containing asoA was cloned and marker-exchange mutagenesis using a kanamycin-resistance cassette was performed to generate an asoA mutation in the low-virulence strain A449L. When analysed by electron microscopy, the mutant A449L-MB exhibited an altered surface morphology. Strands and blebs of membranous material were observed protruding from the disorganized cell surface. This material was shown to contain lipopolysaccharide and A-layer subunit protein. The disorganization of the surface of A449L-MB had no apparent effect on virulence when the bacteria were administered to rainbow trout (Oncorhynchus mykiss) by bath immersion. However, when administered by intraperitoneal injection, the mutant A449L-MB was found to exhibit significantly increased virulence. The predicted amino acid sequence of AsoA shows homology to a number of polytopic membrane proteins involved in translocation across the cytoplasmic membrane.

Electron microscopic examination of factors influencing the expression of filamentous surface structures on clinical and environmental isolates of Aeromonas veronii Biotype sobria.

Strains of Aeromonas veronii biotype sobria isolated from clinical and environmental sources were examined for their expression of surface structures under a variety of culture conditions. When grown on solid media at 37 degrees C, more than 95% of bacteria from the majority of strains isolated from human diarrheal feces and chicken carcasses were non-piliated or expressed only a few pili of long, flexible morphology per cell. Strains isolated from water or other foods were much more likely to express pili. Heavily piliated strains (all sources) possessed pili of several morphological types, including long, flexible pili of varying widths and rigid pili of varying lengths. Expression of Pili was favored by growth at temperatures ca. 20 degrees C and below and growth in liquid medium. Most fecal strains expressed some pili under these conditions. In addition, other surface structures (fibrillar aggregates, fibrillar networks bundle-forming pili) were seen on some strains from most sources. These were also seen most frequently when bacteria were grown in liquid media at temperatures ca. 20 degrees C and below. Pili expression was not dramatically influenced by growth under anaerobic conditions, or in iron-depleted media, or by combinations of the above conditions. The role of the above surface structures in Aeromonas pathogenicity remains to be elucidated.

Temperature-dependent protein and lipopolysaccharide expression in clinical Aeromonas isolates.

Clinical isolates of Aeromonas were grown at eight different temperatures from 10 degrees C to 40 degrees C. Whole cell lysates were examined by SDS-PAGE and major temperature-dependent changes to both protein and lipopolysaccharide (LPS) profiles were identified. Cells grown at the higher temperatures (37 degrees C and 40 degrees C) produced abundantly a protein of c. 60 kDa which was not detected at the lower temperatures. Temperature-dependent expressions of other proteins were also noted but these were more variable among the isolates. An effect of temperature on expression of lipopolysaccharides was also noted in that some strains produced significantly less O-polysaccharides at the higher temperatures. After fractionation of cells, major differences in the expression of cell envelope and outer-membrane proteins between cells grown at low and high temperatures were noted although no unifying patterns could be discerned. Such growth temperature-induced changes in the cell envelope constituents have not been described previously for Aeromonas isolates from man.

Aeromonas salmonicida resistance to complement-mediated killing.

The resistance of Aeromonas salmonicida to complement-mediated killing was investigated by using different strains and their isogenic mutants that had been previously characterized for their surface components. We found that the classical complement pathway is involved in serum killing of susceptible A. salmonicida strains, while the alternative complement pathway seems not to be involved. All of the A. salmonicida strains are able to activate complement, but the smooth strains (with or without the A-layer) are resistant to complement-mediated killing. The reasons for this resistance are that C3b may be bound far from the cell membrane and that it is rapidly degraded; therefore, the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants are serum sensitive because they bind more C3b than the smooth strains, and if C3b is not completely degraded, then the lytic complex (C5b-9) is formed.

Phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.): an ultrastructural study.

BACKGROUND: The ultrastructure of the phagocytic process in fish has not been established in spite of the significant morphofunctional differences detected in the fish immune system with respect to the basic immunological pattern in vertebrates. We report the ultrastructure of the bacterial phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.). METHODS: Head-kidney, blood, and peritoneal exudate leukocytes were challenged with Aeromonas salmonicida and Escherichia coli and processed for transmission electron microscopic study. RESULTS: Macrophages challenged with bacteria showed changes in the cell outline, in the chromatin pattern, and in the ultrastructural features of the cytoplasm as a consequence of an activation process. The phagocytic process consists of the following: 1) Bacteria-macrophage contact. One or more spot contacts between the bacterial wall and the phagocyte membrane are observed. 2) Bacteria engulfment. Slight depressions, membrane invaginations, or cytoplasmic processes are formed at the phagocyte surface. Macrophage processes occasionally surround the bacteria, overlapping and roaming parallel, or a single, long pseudopod encircles a bacterium several times. 3) Endocytic vesicle formation. Macrophages show one or more bacteria inside membrane-bound cytoplasmic vesicles. 4) Phagolysosome formation. Some dense granules (lysosomes) fuse with the endocytic vesicle. 5) Intracellular killing/digestion. Bacteria inside the endocytic vesicles are observed both virtually intact or damaged at different digestion stages. CONCLUSIONS: Sea bass macrophages possess the mechanisms necessary to both engulf and kill bacteria. Cellular and subcellular events in the morphology of phagocytosis and lysosomal dissolution of bacteria fit the general pattern described for mammals.

Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation.

S-layers are paracrystalline protein multimers that cover the entire cell surface of many bacterial species. The presence of an S-layer in Aeromonas salmonicida (also known as A-layer) predisposed this bacterium to apparently unrelated physiological consequences: inhibition of growth at 30 degrees C, enhanced cell filamentation at 37 degrees C, and enhanced uptake of the hydrophobic antibiotics streptonigrin and chloramphenicol. Growth inhibition or enhanced filamentation was not observed when the native A-layer was missing or its arrangement altered, as in Ca(2+)-limited or Ca(2+)- and Mg(2+)-limited cells, in A-layer-negative (A-) cells with an artificially reconstituted A-layer, or in mutants unable to correctly assemble this layer. A-layer-positive cells (A+) were far more sensitive to the intracellularly acting antibiotics streptonigrin and chloramphenicol than were A- cells, and streptonigrin-resistant mutants were predominantly A-. Hemin, a compound known to specifically bind to the A-layer, alleviated streptonigrin toxicity to A+, but not A-, cells. As well, Ca(2+)- and Mg(2+)-limited cells, or mutants harboring A-layer defects had a reduced sensitivity to streptonigrin, and A- cells with reconstituted A-layers remained resistant to streptonigrin and chloramphenicol. Thus, the presence of a native A-layer arrangement on the cell surface, and not the mere presence of A-layer protein subunits, predisposed A. salmonicida toward the aforementioned physiological consequences. The A-layer is suggested to specifically effect these consequences, in particular the permeation of streptonigrin or chloramphenicol, by a specific interaction of A-layer subunits with the outer membrane.

Adhesion to and invasion of human colon carcinoma Caco-2 cells by Aeromonas strains.

The enteropathogenicity of Aeromonas strains that showed mannose-resistant adhesion to INT407 cells was evaluated by infecting Caco-2 cells and observing them by light and electronmicroscopy. Five of six strains adhered in large numbers to Caco-2 cells in the presence of mannose and caused cytopathic effects. Two strains of Aeromonas spp. seemed to invade Caco-2 cells, as membrane-bound bacteria were seen within the cytoplasm of these cells; however, staining by acridine orange-crystal violet appeared to show intracellular fluorescent bacteria in three strains. Fimbriae did not appear to play an important role in adhesion because fimbrial structures were not seen by transmission electronmicroscopy. Adhesion of four strains was inhibited by the addition of L-fucose. The strains were negative in the fluorescence actin staining test, which in enteropathogenic Escherichia coli strains correlates with the ability to attach and efface intestinal microvilli. The DNA of the Aeromonas strains did not hybridise with the E. coli eae and ipaB probes, associated with attaching and effacing ability and invasion, respectively. These results give support to the enteropathogenicity of adhesive strains of Aeromonas spp., although the mechanisms of adhesion, and possibly invasion, remain to be elucidated.