Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

Interaction between Aeromonas veronii and epithelial cells of spotted sand bass (Paralabrax maculatofasciatus) in culture

An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms). Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease. Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer. veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates. The three primary-culture cells are susceptible to Aer. veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial cells. Aer. veronii adherence was dependent on bacterial load and incubation time. The effect of glycoconjugates on Aer. veronii adhesion was investigated by pre-incubating Aer. veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides. In addition, the participation of a 48-kDa Aer. veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer. veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer. veronii MCBP. Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer. veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.     

Adhesive properties of a LamB-like outer-membrane protein and its contribution to Aeromonas veronii adhesion

AIMS: To identify and characterize nonfimbrial proteins from Aeromonas veronii involved in the attachment to epithelial cells in vitro. METHODS AND RESULTS: Two Aer. veronii mucin- and lactoferrin-binding proteins with molecular masses of 37 and 48 kDa were identified by Western blot analysis. According to its N-terminal amino acid sequence, the 48-kDa protein was identified as Omp48, an outer-membrane protein similar to LamB of Escherichia coli. LamB is a well-known porin involved in maltose transport across the outer membrane in E. coli. In a microtitre plate assay, Omp48 bound to the immobilized extracellular matrix proteins collagen and fibronectin, and the mucin- and lactoferrin-binding activity was confirmed. Adhesion of Omp48 to mucin, lactoferrin and collagen was diminished by preincubation with homologous glycoproteins or other carbohydrates, suggesting a putative Omp48 lectin-like binding domain. Anti-Omp48 antiserum significantly inhibited the Aer. veronii adhesion to confluent HeLa cell monolayers and pretreatment of cells with purified Omp48 elicited competitive inhibition of adhesion. Similarly, cross-inhibition of Aer. hydrophila and Aer. caviae adhesion was achieved with the same treatments, indicating the existence of a conserved surface protein among these species. CONCLUSIONS: Taken together, these data indicate that Omp48 is involved in Aer. veronii adhesion to epithelial cells and might be an alternative adhesion factor of this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesive potential of Aeromonas spp. is correlated with pathogenicity; however, the adhesion mechanism is complex and not well understood. This study provides evidence of a putative adhesion factor that might be contributing to pathogenicity of Aer. veronii and could be used for vaccine development.     

Incidence of Aeromonas species in influent and effluent of urban wastewater purification plants

Influent and effluent samples from urban wastewater plants were examined for presumptive Aeromonas. Removal efficiency was comparatively low for both presumptive Aeromonas (96.5%) and faecal coliforms (95.8%). Starch ampicillin agar (SA) medium was superior to mA medium for selectivity, with 32.3% of typical colonies confirmed as Aeromonas spp., and for the recovery of Aer. veronii biotype veronii.     

Differential regulation of caspase-1 activation via NLRP3/NLRC4 inflammasomes mediated by aerolysin and type III secretion system during Aeromonas veronii infection

Aeromonas spp. are Gram-negative bacteria that cause serious infectious disease in humans. Such bacteria have been shown to induce apoptosis in infected macrophages, yet the host responses triggered by macrophage death are largely unknown. In this study, we demonstrate that the infection of mouse bone marrow-derived macrophages with Aeromonas veronii biotype sobria triggers activation of caspase-1 with the ensuing release of IL-1β and pyroptosis. Caspase-1 activation in response to A. veronii infection requires the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain and both the NLRP3 and NLRC4 inflammasomes. Furthermore, caspase-1 activation requires aerolysin and a functional type III secretion system in A. veronii. Aerolysin-inducing caspase-1 activation is mediated through the NLRP3 inflammasome, with aerolysin-mediated cell death being largely dependent on the NLRP3 inflammasome. In contrast, the type III secretion system activates both the NLRP3 and NLRC4 inflammasomes. Inflammasome-mediated caspase-1 activation is also involved in host defenses against systemic A. veronii infection in mice. Our results indicated that multiple factors from both the bacteria and the host play a role in eliciting caspase-1 activation during A. veronii infection.     

Phenotypic study by numerical taxonomy of strains belonging to the genus Aeromonas

AIMS: This study was undertaken to cluster and identify a large collection of Aeromonas strains. METHODS AND RESULTS: Numerical taxonomy was used to analyse phenotypic data obtained on 54 new isolates taken from water, fish, snails, sputum and 99 type and reference strains. Each strain was tested for 121 characters but only the data for 71 were analysed using the 'SSM' and 'SJ' coefficients, and the UPGMA clustering algorithm. At SJ values of > or = 81.6% the strains clustered into 22 phenons which were identified as Aer. jandaei, Aer. hydrophila, Aer. encheleia, Aer. veronii biogroup veronii, Aer. trota, Aer. caviae, Aer. eucrenophila, Aer. ichthiosmia, Aer. sobria, Aer. allosaccharophila, Aer. media, Aer. schubertii and Aer. salmonicida. The species Aer. veronii biogroup sobria was represented by several clusters which formed two phenotypic cores, the first related to reference strain CECT 4246 and the second related to CECT 4835. A good correlation was generally observed among this phenotypic clustering and previous genomic and phylogenetic data. In addition, three new phenotypic groups were found, which may represent new Aeromonas species. CONCLUSIONS: The phenetic approach was found to be a necessary tool to delimitate and identify the Aeromonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable traits for identifying Aeromonas as well as the possible existence of new Aeromonas species or biotypes are indicated.     

Molecular cloning,sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii

AIMS: To clone, sequence and characterize the gene encoding the Omp48, a major outer membrane protein from Aeromonas veronii. METHODS AND RESULTS: A genomic library of Aer. veronii was constructed and screened to detect omp48 gene sequences, but no positive clones were identified, even under low stringency conditions. The cloned gene probably was toxic to the host Escherichia coli strain, so the cloning of omp48 was achieved by inverse PCR. The nucleotide sequence of omp48 consisted of an open reading frame of 1278 base pairs. The predicted primary protein is composed of 426 amino acids, with a 25-amino-acid signal peptide and common Ala-X-Ala cleavage site. The mature protein is composed of 401 amino acids with a molecular mass of 44,256 Da. CONCLUSIONS: The omp48 gene from Aer. veronii was cloned, sequenced and characterized in detail. BLAST analysis of Omp48 protein showed sequence similarity (over 50%) to the LamB porin family from other pathogenic Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial diseases are a major economic problem for the fish farming industry. Outer membrane proteins are potentially important vaccine components. The characterization of omp48 gene will allow further investigation of the potential of Omp48 as recombinant or DNA vaccine component to prevent Aer. veronii and related species infections in reared fish.     

Detection of aerolysin gene in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from fish and pond water

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg.     

Assessment of media used for selective isolation of Aeromonas spp.

A. GAVRIEL AND A.J. LAMB. 1995. A group of selective media were evaluated for their ability to support growth and recovery of type species of the mesophilic, motile group from the genus Aeromonas . With both low and high inoculum densities Aer. hydrophila and Aer. caviae grew well on ampicillin dextrin agar, glutamate starch penicillin agar and starch glutamate ampicillin penicillin agar whereas Aer. veronii only grew with a high level of inoculum. Aeromonas sobria and Aer. schubertii displayed little growth on any of these media regardless of the inoculum size. These results indicate that environmental studies attempting to evaluate the relative distribution and abundance of the members of the motile Aeromonas group would not recover all the strains with equal effectiveness using these particular media.     

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae , 46 (22%) as Aer. sobria , 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii . Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.     

Bacteriocin activity and probiotic activity of Aeromonas media

Three strains of Aeromonas media (161, A164 and A199) were shown to be active in-vitro producers of bacteriocin-like inhibitory substances (BLIS). For example, the producer strain, Aer. media A199, displayed antagonistic activity against all strains tested of Aer. caviae, Aer. hydrophila, Aer. salmonicida, Aer. veronii var. sobria, Listonella anguillarum, Photobacterium damsella, eight species of Vibrio and Yersinia ruckeri. Because of this wide-ranging activity against fish/shellfish pathogens, A199 was chosen for the probiotic work. By contrast, however, the BLIS produced by A199 did not inhibit the growth of Enterococcus seriolicida. The aim of the project was to ascertain whether or not the activity observed in vitro could be repeated in vivo. The ability of BLIS-producing strain A199 to act as a probiotic was assessed on the host animal, Crassostrea gigas, by testing whether or not strain A199 could prevent death of the oyster larvae when challenged with V. tubiashit. Whereas larvae, challenged with the Vibrio, died within 5 days, the presence of both the pathogen and the probiotic strain, together, did not affect the viability of the larvae over the same time period; the viability of larvae challenged with A199 alone was also unaffected when compared with the viability of unchallenged larvae (controls). These findings have important, economic implications for those engaged in the oyster producing industry where heavy losses can be experienced as a result of an infectious outbreak. At this stage, the association between BLIS activity and probiotic activity is circumstantial and, hence, future work will involve the use of non-BLIS-producing strains of Aer. media and BLIS-negative variants of the producer. Moreover, extension of the project will involve the use of other BLIS-producing strains (A161, and A164), hosts (salmon, crayfish, scallops and abalone) and pathogens.     

Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29

Photorhabdus sp. strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures. The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism. The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase. Protease secretion was higher in cultures growing at lower temperatures. At 10 degrees C the activity was highest and remained constant for over 7 days, whereas at 23 and 28 degrees C it showed a steady decrease. Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography. PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50 degrees C. Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14. Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence. In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B. PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA. Its activity ranged between 10 and 80 degrees C and was optimal at pH 7 and 50 degrees C. PrtS also destroyed insect antibacterial factors. Three fragments of PrtS showed homology with a putative metalloprotease of P. luminescens TTO1. Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules.     

Changes in particulate-bound protease activity during cold acclimation in Tetrahymena pyriformis

The protease activity, as assayed at pH 8.0 with azocasein as substrate, of a ciliate protozoan Tetrahymena pyriformis NT-1, was found to alter by growing the cells at various constant temperatures or at shifted temperatures. The intracellular protease activity, when cells were grown at either constant 39 degrees C or 15 degrees C, was decreased throughout the growth phase with significant secretion into the medium. On the other hand, when the culture temperature was transferred from 39 degrees C to 15 degrees C, the protease activity in cells was greatly increased up to about 28-fold at 8 h after the shift. There was, however, no secretion into the medium during the cold acclimation after the shift, where no cell division occurred. The elevated protease activity was quickly decreased to the control level when the culture was warmed to 39 degrees C after 8-h chilling, and recovery of normal cell division was seen. The marked increase in the protease activity caused by the shift to 15 degrees C was completely blocked by the addition of either cycloheximide or actinomycin D. The thermally induced enhancement of protease activity was found to occur with no different preference between three protease fractions.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.     

致病性维罗纳气单胞菌检测方法的建立

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点,建立能够检测致病性维罗纳气单胞菌的PCR检测方法.通过序列比对分析气单胞菌的16S rRNA基因序列,筛选对维罗纳气单胞菌特异的引物,用于检测种特异性,利用气单胞菌气溶素基因保守引物,检测菌株的致病性,并进行反应条件和反应体系的优化,灵敏度试验和特异性试验.发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法,对12株气单胞菌和10株非气单胞菌的检测结果显示,所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带,而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带,其它菌株都不能扩增到目的条带.灵敏度试验表明,该反应体系的检测灵敏度为1.35×10-3 mg/L.我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌,并具有高度灵敏性.     

Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates

M. DORSCH, N.J. ASHBOLT, P.T. COX AND A.E. GOODMAN. 1994. Published 16S rDNA sequencing data for Aeromonas species were analysed and the validity of signature sequences derived from our investigations of these sequences was examined by sequencing the corresponding 16S rDNA regions of 67 environmental isolates from sewage effluents and receiving waters around Sydney and one clinical isolate, all previously classified as Aeromonas species. Species-specific probes for Aer. hydrophila and Aer. veronii were designed and tested in PCR assays and clearly discriminated these species from the other Aeromonas isolates as identified by 16S rDNA sequences.