Mucosal immune response of spotted sand bass Paralabrax maculatofasciatus (Steindachner, 1868) orally immunised with an extracellular lectin of Aeromonas veronii

To assess the immunogenic and immunoprotective role of the extracellular lectin from Aeromonas veronii (MCBP), which has affinity for mucosal constituents such as mucin, lactoferrin, immunoglobulins and collagen, spotted sand bass (Paralabrax maculatofasciatus) were orally immunised either with soluble MCBP, adjuvant-conjugated MCBP or immobilised MCBP on latex microspheres. The results suggest that the MCBP is capable of eliciting protective immunity against A. veronii infections when administered orally. The highest mucosal immune response was elicited in fish immunised with MCBP covalently linked to cholera toxin B subunit (CTB) or to Escherichia coli heat-labile toxin (hLT). MCBP-CTB was found to elicit immunoprotection against a challenge with live Aeromonas cells with a relative percent survival of almost 70% and without the expression of the severe histopathological alterations induced by A. veronii.     

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.     

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.     

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

AIMS: To investigate the prevalence of Aeromonas in a major waterway in South East Queensland, Australia, and their interactions with a gut epithelial model using Caco-2 cells. METHODS AND RESULTS: A total of 81 Aeromonas isolates, collected from a major waterway in South East Queensland, Australia, were typed using a metabolic fingerprinting method, and tested for their adhesion to HEp-2 and Caco-2 cells and for cytotoxin production on Vero cells and Caco-2 cells. Aeromonas hydrophila had the highest (43%) and Aeromonas veronii biovar sobria had the lowest (25%) prevalence. Four patterns of adhesion were observed on both HEp-2 and Caco-2 cell lines. Representative isolates having different phenopathotypes (nine strains) together with two clinical isolates were tested for their translocation ability and for the presence of virulence genes associated with pathogenic Escherichia coli. The rate and degree of translocation across Caco-2 monolayers varied among strains and was more pronounced with LogA pattern. Translocation was associated with the adherence of strains to Caco-2 cells microvilli, followed by internalization into Caco-2 cells. Two Aer. veronii biovar sobria strains were positive for the presence of heat-labile toxin genes, with one strain also positive for Shiga-like toxin gene. CONCLUSIONS: Pathogenic strains of Aeromonas carrying one or more virulence characteristics are highly prevalent in the waterways studied and are capable of translocating across a human enterocyte cell model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that Aeromonas strains carrying one or more virulence properties are prevalent in local waterways and are capable of translocating in a human enterocyte cell culture model. However, their importance in human gastrointestinal disease has yet to be verified under competitive conditions of the gut.     

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.     

Anti-adhesive activity of sulphated exopolysaccharides of microalgae on attachment of red sore disease-associated bacteria and helicobacter pylori to tissue culture cells

Because of the affinity of certain bacterial species for sulphated glycoconjugates exposed on the epithelial cells of susceptible hosts, we hypothesized that sulphated exopolysaccharides of microalgae can be used in anti-adhesive therapies against bacterial infections, both in cold- and warm-blooded animals. In this study we found that adhesion of the human pathogen Helicobacter pylori to the HeLa S3 cell line, and adhesion of the fish pathogens Vibrio campbellii, V. ordalii, Streptococcus saprophyticus, and Aeromonas veronii to spotted sand bass primary tissue culture cells, can be effectively blocked with the various sulphated exopolysaccharides used.     

Molecular characterization and distribution of virulence-associated genes amongst Aeromonas isolates from Libya

AIMS: The aim of the study was to type 52 Aeromonas spp. isolates from chicken carcasses, children with diarrhoea and a hospital environment in Libya, and to determine the distribution of putative virulence genes amongst them. METHODS AND RESULTS: Macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S rRNA and aroA genes were used to type the isolates. Whereas 30 of 32 chicken isolates were identified as Aeromonas veronii, eight of 12 environmental isolates were Aer. caviae. Three species were identified amongst the eight isolates from children. Aeromonas veronii and Aer. caviae isolates could be divided into eight and five PFGE types, respectively. All species could be further subtyped into one of 21 aroA PCR-RFLP groups. Aerolysin-like haemolysin or enterotoxin gene sequences were detected in all the isolates. Overall carriage rates for hlyA and alt were 77 and 75%, respectively. CONCLUSIONS: Seven of eight isolates from children were of different subtypes, indicating a lack of any common source of acquisition. Isolates of common molecular type did not share identical distributions of putative virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the effectiveness of using molecular typing to identify and study genetic variation amongst Aeromonas isolates.     

Distribution of Aeromonas spp. as identified by 16S rDNA restriction fragment length polymorphism analysis in a trout farm

AIMS: This study used restriction fragment length polymorphism (RFLP) with Aeromonas-specific primers to identify species of Aeromonas and to investigate their distribution in a trout farm and stream. METHODS AND RESULTS: In January, May, August and November 2000, presumptive Aeromonas species were recovered from a farm and a sedimentation pond in a fish farm and stream, and identified by PCR-RFLP analysis with Aeromonas-specific primers. The specificity of Aeromonas-specific primers and the suitability of PCR-RFLP analysis for identifying Aeromonas spp. were confirmed with fatty acid methyl esters (FAMEs) and 16S rDNA sequencing analyses, respectively. Levels of Aeromonas spp. sampled in May and August were higher than in January and November at all sampling sites. Aeromonas salmonicida was the dominant species in January and November, and the proportion of pathogenic species (Aer. hydrophila, Aer. caviae and Aer. veronii) increased in May and August. CONCLUSIONS: PCR-RFLP analysis with Aeromonas-specific primers is a rapid and reliable method for identifying widely distributed Aeromonas spp. from environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To minimize human health risk, monitoring the levels and species composition of Aeromonas in fish farm is advisable.     

Production and secretion of collagen-binding proteins from Aeromonas veronii

Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction. The results of this study show that 98% of the total CNBP produced by Aer. veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane. CNBP is specifically secreted from Aer. veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins. CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C. The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase. There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium. CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer. hydrophila. It is concluded that CNBP secretion from Aer. veronii must be achieved by a mechanism different from that reported for aerolysin secretion.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

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.     

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.     

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.     

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

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.     

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.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract

Aims: In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion. Methods and Results: We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25°C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI-ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11. Conclusion: The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract. Significance and Impact of the Study: We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract.     

广东中山地区气单胞菌环境株的分离、鉴定及系统发生关系

从广东省中山市的池塘水样、底泥、健康鱼、肠道及稻田土样中用Aeromonas的选择培养基分离到10株气单胞菌。通过生理生化测试、16S rDNA序列测定、与气单胞菌典型菌株的16S rDNA序列进行比对和聚类分析,对它们进行了鉴定,并研究了它们之间的系统发生关系。结果显示该地区环境中气单胞菌的优势种除A.hydrophila(HG1组)外,还有A.caviae(HG4组)、A.jandaei(HG9组)和A.veronii(HG10组),其中后两种是国内新记录。这是国内首次对环境中气单胞菌多样性进行研究。     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.