ENTEROPATHOGENIC AEROMONADS

Hin-chung Wong

Department of Microbiology Soochow University

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1. INTRODUCTION 2. OCCURRENCE IN ENVIRONMENT AND CLINICAL SPECIMEN 3. CHARACTERISTICS AND TAXONOMY

4. TYPING 4.1. PhenePlate rapid screening system 4.2. FAME and AFLP analysis 4.3. OMP Profiles and Restriction endonuclease analysis 4.4. Ribotyping 4.5. Pulsed-field gel electrophoresis 4.6. Typing with PCR techniques 4.7. Sequences encoding lipase and enterotoxin genes

5. ISOLATION AND ENUMERATION 6. VIRULENCE FACTORS 6.1. Invasion 6.2. Adhesion 6.3. Hemagglutination 6.4. Autoagglutination 6.5. Hydrophobicity 6.6. Hemolysins and Enterotoxins 6.6.1. Cross-Reactive to Cholera Toxin 6.6.2. Non-cross-reactive to Cholera Toxin 6.6.3. Heat-stable enterotoxins 6.7. Activities of Aeromonas toxins

6.8. DNA adenine methyltransferase 6.9. Quorum sensing 6.10. Type III secretion system 6.11. Alternative host model

7. CONCLUSION 8. REFERENCES ___________________________________________________________

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1. INTRODUCTION Aeromonas strains are common pathogens in aquaculture. Some of these strains have been involved in human gastroenteritis. However, the role of this Genus as a foodborne pathogen is still not clear. Aeromonas strains were isolated from feces of 21 of 561 (3.7%) children with gastroenteritis and 12 of 576 (2.1%) children without intestinal disturbances (control), but the difference is not significant statistically (Figura et al., 1986). According to clinical survey, the frequency of A. hydrophila cases during warm months was 1.25 case per month, whereas during cold months it was 0.83 case per month. Although much less common than Campylobacter jejuni, which was found in 8.3% of diarrhea cases, A. hydrophila was only slightly less frequent than Salmonella sp. isolates, which were found in 1.6% of diarrhea cases (Fig. 1) (Agger et al., 1985).

2. OCCURRENCE IN ENVIRONMENT AND CLINICAL SPECIMEN Most studies involving gastroenteritis caused by A. hydrophila have concentrated on its transmission in contaminated water supplied. Motile Aeromonas species occur widely in water, sludge and sewage (Kaper et al., 1981). Population of Aeromonas in the environment is controlled by seasonal factors

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(temperature, solar radiation, phytoplankton) (Monfort and Baleux, 1990). In the environment, Aeromonas are sometimes isolated more frequently than E. coli, however, the survival capability of A. hydrophila is similar to E. coli (Chung and Yu, 1990). Aeromonas species are potential food-poisoning agent. A. hydrophila is psychrotrophic and has been associated with the spoilage of refrigerated (5C) animal products including chicken, beef, pork, lamb, fish, oysters, crab, and milk (Majeed et al., 1989). Aeromonas may also produce toxins at low temperature under suitable growth conditions (Krovacek et al., 1991; Majeed et al., 1989). Aeromonas were isolated from all the produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce (range 1.0 x 102 to 2.3 x 104 CFU/g). The count of Aeromonas increased 10- to 1,000-fold during 2 weeks of storage at 5C (Callistor and Agger, 1987). All the food samples surveyed (included red meats, chicken, raw milk and seafood) contained with A. hydrophila, and the count at the time of purchase ranged from 1x102/g to 5x105/g (Palumbo et al., 1985). In Denmark, Aeromonas occurred in 28% of drinking water, 28% of ice cream and 10% of mayonnaise salads (Kn:chel and Jeppesen, 1990). In a survey by Callister and Agger (Callistor and Agger, 1987), all produce sampled contained with Aeromonas sp., and 92% of the 12 kinds of produce yielded cytotoxic Aeromonas sp. All the A. hydrophila isolates and 6% of the A. caviae were cytotoxic, and 90% of cytotoxic Aeromonas strains produced hemolysins (Callistor and Agger, 1987).

The persistence and transmission of Aeromonas in a duckweed aquaculture-based hospital sewage water treatment plant in Bangladesh was studied. Aeromonas was found at all sites of the treatment plant, in 40% of the samples from environmental control ponds, in 8.5% of the samples from hospitalized children suffering from diarrhea, and in 3.5% of samples from healthy humans. A significantly high number of Aeromonas bacteria was found in duckweed, which indicates that duckweed may serve as a reservoir for these bacteria. All tested isolates of the major types were positive for the cytolytic enterotoxin gene (Rahman et al., 2007). Toxigenicity of A. hydrophila isolates from various seafoods in Taiwan was assayed, and 79.2%, 91.7%, and 81.3% or these isolates produced hemolysin, cytotoxin and protease, respectively (Tsai and Chen, 1995).

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High incidence of aeromonads occurs in food (Neyts et al., 2000). High toxigenic isolates are present in environmental samples. Of the clinical isolates of Aeromonas, 29.4% were enterotoxigenic, 43.1% were hemolytic and 89% were cytotoxigenic. Among the food isolates, 18.2% were enterotoxigenic, 17.1% were hemolytic and 72.7% were cytotoxigenic. Aeromonas sobria and A. veronii produced more enterotoxin and cytotoxin than the other isolates, whereas A. veronii and A.salmonicida produced cell-free hemolysin (Martins et al., 2002). 3. CHARACTERISTICS AND TAXONOMY Aeromonas are Gram negative rods with rounded ends to coccoid, 0.3-1.0 μm in diameter and 1.0-3.5 μm in length, occur singly, in pairs or short chains. This Genus is divided into two well separated groups: the nonmotile A. salmonicida and the motile group. A. salmonicida cells are nonmotile and atrichous. Optimum growth temperature is 22-25C. Most strains grow at 5C (Beuchat, 1991). Maximum temperature for growth is usually 35C. The following carbohydrates are usually fermented by A. salmonicida: arabinose, trehalose, galactose, mannose and dextrin. The following biochemical tests are universally negative for A. salmonicida: growth in KCN broth, growth in nutrient broth containing 7.5% NaCl, urease, ornithine decarboxylase, tetrathionate reductase and acidification of media containing rhamnose, sorbose, sorbitol, lactose, raffinose and cellobiose. Some strains produce pigments. The motile Aeromonas can be divided into three species: A. hydrophila, A. caviae, and A. sobria. The optimum growth temperature for motile Aeromonas species is 28C. Some strains can grow at 5C. The maximum growth temperature is usually 38-41C. Temperature-dependent differences in soluble protein production in some of the Aeromonas strains were observed (Statner et al., 1988). On nutrient agar, colonies are round, raised, with an entire edge and a smooth surface, translucent and white to buff (deep yellow) in color. Usually most strains do not have pigment. The following tests are universally positive for motile species: catalase, starch hydrolysis, lecithinase, phosphatase, arginine dihydrolase, hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG), growth in nutrient broth without NaCl, and fermentation of mannitol, trehalose, fructose, galactose and dextrin. Characteristics of the Aeromonas species are listed in Table 1

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(Popoff, 1984). Eleven ornithine positive strains of Aeromonas received by the Center for Disease, USA, were studied by DNA hybridization tests, biochemical analyses, and antimicrobial susceptibility tests and nine of them named as A. veronii and 2 still classified as Aeromonas species ornithine positive (Stelma, Jr. et al., 1988).

A. salmonicida is a serologically homogeneous species. Motile Aeromonas species contain 12 O antigens and 9 H antigens. In naturally occurring A. salmonicida strain plasmids conferring resistance to streptomycin, chloramphenicol, tetracycline and sulfathiazole. The R factors also present in the motile Aeromonas. Most clinical Aeromonas isolates of Saudi Arabia were inhibited by gentamicin, amikacin, chloramphenicol and tetracycline and resistant to broadspectrum penicillins and many were also resistant to cefuroxime and cefoxitin (Gosling, 1986). Enterotoxigenic activity of Aeromonas seems to be related to biotyping. Of 131 enterotoxigenic strains, 120 were VP positive and hydrolyzed arabinose. Of 43 non-enterotoxigenic strains, 19 were VP positive and hydrolyzed arabinose (Burke et al., 1982). But in another paper, some enterotoxigenic strains are VP positive, arabinose negative, and positive lysine decarboxylase (Kirov et al., 1986). Cumberbatch et al. (Cumberbatch et al., 1979) reported that cytotoxin production correlated with a positive lysine decarboxylase phenotype (98%) or a positive VP phenotype (94%), compared to 27% lysine decarboxylase-positive and 23% VP positive of the cytotoxin-negative isolates. The enterotoxigenicity of

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Aeromonas isolated from fish is significantly related with lysine decarboxylase among many properties examined (Santos et al., 1988). In another paper, Burke et al. reported that fermentation of arabinose occurred with 58.8% of the environmental strains and 15% of the clinical isolates; 39.4% of the strains from water and 6.8% of the fecal isolates fermented salicin (Burke et al., 1984).

Cluster analysis of the phenotypes of clinical and environmental Aeromonas strains revealed three major phenons equivalent to the A. hydrophila, A. caviae, and A. sobria groups, each of which contained more than one genospecies and more than one named species based on DNA hybridization (Table ) (Altwegg et al., 1990).

4. TYPING

4.1. PhenePlate rapid screening system

In total, 1,315 presumptive Aeromonas isolates were biochemically typed by the PhenePlate rapid screening system (PhP-AE). A selection of 90 representative isolates was further analyzed with PhenePlate (PhP) extended typing (PhP-48), fatty acid methyl ester analysis, and amplified fragment length polymorphism (AFLP) fingerprinting. In addition, the prevalence of the putative virulence factors hemolysin and cytotoxin and the presence of the cytolytic enterotoxin gene (AHCYTOEN) were analyzed (Rahman et al., 2007). 4.1 Biochemical fingerprinting with the PhP system.

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The isolates can be typed by a biochemical phenotyping method, the PhP-AE system (PhPlate Microplate Techniques AB, Stockholm, Sweden) (Rahman et al., 2007). The PhP fingerprints of the isolates ae compared pairwise, and the similarity between each pair is expressed as a correlation coefficient. Isolates are assigned to the same PhP type when they showed a correlation coefficient higher then 0.975. The diversities among populations are calculated as Simpson's diversity index (Di), and clustering of the isolated is performed.

4.2. FAME and AFLP analysis

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Aeromonas strains are identified to the genomic species level by using gas-liquid chromatographic analysis of cellular fatty acid methyl esters (FAMEs). Isolates can be subjected to whole-genome fingerprinting using amplified fragment length polymorphism (AFLP) analysis. The AFLP profiles of unknown isolates are compared with the laboratory-based identification library AEROLIB, comprising AFLP profiles generated from a collection of well-characterized type and reference strains encompassing all currently recognized Aeromonas taxa (Huys et al., 1996). 4.3. OMP Profiles and Restriction endonuclease analysis The outer membrane protein (OMP) composition (OMP typing) of fecal Aeromonas strains from hybridization groups (HGs) 1 (A. hydrophila), 4 (A. caviae), and 8 (A. veronii) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a phenotypic typing method. Almost every isolate of HG-1 and HG-8 had a unique OMP profile, in contrast to isolates of HG-4, which were separated into five different OMP types. It was possible to recognize HGs 1, 4, and 8 by OMP profiles (Fig. ) (Kuijper et al., 1989).

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Aeromonas strains from HGs 1, 4, and 8 were tested by whole-cell DNA restriction endonuclease analysis (REA) as a genetic typing method. All strains tested by REA (with SmaI) had different DNA digestion patterns. Although additional DNA-rRNA hybridization analyses with SmaI and 16S and 23S rRNAs from E. coli showed a reduction in the number of restriction bands to 8 to 13 hybridized fragments (Fig. ), the discriminative value was less when compared with that obtained by REA (Kuijper et al., 1989).

4.4. Ribotyping

Ribotyping for the differentiation of aeromonads isolated from patients with gastroenteritis and from the source water, treatment plant, and distribution system of a small public water supply was performed. Ribotyping patterns of aeromonads recovered from well 1, detention basin, sand filter, softener, and distribution samples were compared with those of the five clinical isolates. All patient strains were unique; however, identical ribotypes of A. hydrophila and A. sobria isolated from multiple sites in the water system indicated colonization of a well, sand filters, and the softener, with the potential for sporadic contamination of distribution water (Moyer et al., 1992).

Ribotyping was used to study the epidemiology of Aeromonas associated

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gastro-enteritis in young children. Ribotyping patterns of Aeromonas strains (A. caviae, A. hydrophila, A. eucrenophila, A. veronii, and A. encheleia) isolated from primary stool cultures of sick children were compared using the GelCompare software with patterns of 104 strains isolated from their household environment in order to investigate the route of transmission of these bacteria. Fifteen strains (approximately 47%) isolated from stool cultures of patients showed the same riboprofile as strains found in contacts or environment. In particular, three strains isolated from patients shared the same riboprofile with strains found in their domestic environment (Demarta et al., 2000).

Ribotyping has been used to type A. salmonicida. Most epidemiologically

unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical (Pedersen et al., 1996). 4.5. Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis was developed to analysis a total of 103 isolates of Aeromonas spp. obtained from a natural mineral water and from surface streams. Genomic DNA was digested with XbaI. All A. caviae isolates from the natural mineral water belonged to the same clone, and an analogous clonal identity was found among A. hydrophila isolates. Aeromonas isolates from surface waters showed high molecular heterogeneity and were not related to the clones found in the natural mineral water (Fig. ). The presence of aeromonads chronically found in the natural mineral water was a likely consequence of a localized development of a biofilm, with no exogenous contamination of the aquifer (Villari et al., 2003).

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4.6. Typing with PCR techniques Random amplified polymorphic DNA (RAPD), REP-PCR and ERIC-PCR

were developed for Aeromonas species. The RAPD method involves the use of short random sequence primers, usually 9 to 10 nucleotides long, and low-stringency primer annealing conditions to amplify arbitrary fragments of template DNA. The single primer anneals anywhere on the genome where a near-complementary sequence exists, and if two priming sites are sufficiently close, PCR then amplifies the fragment between them. The REP-PCR method uses primers complementary to REP elements of bacterial genomic DNA. PCR amplification of template genomic DNA results in products of different sizes, presumably reflects the distance and orientation of endogenous repeats. The ERIC-PCR method utilizes primers complementary to ERIC sequences of bacterial genomic DNA (Szczuka and Kaznowski, 2004).

4.7. Sequences encoding lipase and enterotoxin genes DNA fragments were amplified by PCR from all tested strains of A.

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hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains (Watanabe et al., 2004).

5. ISOLATION AND ENUMERATION There is no widely accepted protocols for the isolation of Aeromonas. Food samples are stored at 5C for 7 days and homogenized in phosphate buffer saline and dilutions could be directly plated onto Starch Ampicillin Agar. The starch-ampicillin agar consists of phenol red agar base (Difco)(31 g), soluble starch (10 g) and distilled water (1 L). Ampicillin is added to achieve a concentration of 10 μg/ml after autoclaving of the medium (Palumbo et al., 1985). After incubation at 28C for 24 h, the plates are flooded with Lugol iodine solution, and amylase positive colonies were scored as presumptive A. hydrophila. A new medium SGAP-10C agar (major components: sodium glutamate, soluble starch, penicillin, ampicillin, glucose) was shown to be more selective and permitted better recovery of stressed Aeromonas (Huguet and Ribas, 1991).

Six selective agents (ampicillin, novobiocin, cephalothin, bile salts, brilliant green and ethanol) were tested during the development of a selective enrichment broth for the isolation of Aeromonas sp. from food. Cephalothin at 10 mg/l was found to be the best selective agent owing to its greater selectivity and efficiency in recovering stressed and lower cell concentrations of Aeromonas sp. Higher concentrations (15-25 mg/l) of cephalothin were inhibitory to some strains of A. sobria. Cephalothin (10 mg/l) was incorporated in buffered dextrin broth (BCDB-10) and alkaline peptone water (CAPW-10) for the isolation of Aeromonas sp. (Sachan and Agarwal, 2000). The isolates are confirmed by the following tests: Gram stain, oxidase, catalase,

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DNase, resistance to vibriostatic agent O/129, resistance to novobiocin, and API 20E strips (Palumbo et al., 1985). Since simple microflora occurs in the clinical samples, selective media useful in clinical isolation may not be good in surveying food samples. Several selective media for isolation of Aeromonas and Plesiomonas from human feces were evaluated, alkaline peptone water, trypticase soy broth with ampicillin, inositol-brilliant green-bile salts agar, dextrin-fuchsin-sulfite agar, xylose- sodium deoxycholate-citrate agar, and Pril-xylose-ampicillin agar were media with optimal sensitivity and specificity for Aeromonas spp. (Table 2, 3) (von and Bucher, 1983). Mishara et al. showed that Sheep blood agar with 30 mg/L of ampiciIlin was the best among media evaluated, and also suggested DNase-toluidine blue agar to isolate Aeromonas that are susceptible to the high ampicillin concentration in the sheep-blood agar (Table 4) (Mishra et al., 1987). Kelly et al. (Kelly et al., 1988) also showed the Ampicillin blood agar (ampicillin, 20 μg/ml) is superior to other media evaluated, but also suggested to use more than one medium.

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6. VIRULENCE FACTORS Aeromonas species cause a variety of diseases in fish, amphibians, and mammals. In humans, Aeromonas strains are common isolates from skin and wound infections in otherwise healthy persons. Aeromonas sepsis, which is frequently fatal in humans, is usually associated with malignancies or other chronic underlying illnesses. Bacteremias have also been reported in persons hospitalized for diarrhea. The three motile Aeromonas spp. are usually isolated from patients (Figura et al., 1986; Gosling, 1986). In Tasmania, Australia, A. sobria comprised 35% of the clinical isolates and 16% of the water isolates, A. hydrophila comprised 56 and 79%, and A. caviae comprised 9 and 5%. A total of 42% of the clinical isolates and 15% of the environmental isolates were enterotoxigenic (by the suckling mouse assay). The majority (74%) of enterotoxigenic isolates were A. sobria (Kirov et al., 1986). In another paper, most of the A. hydrophila and A. sobria were enterotoxigenic. Burke et al. reported that 91% of clinical isolates and 70.2% of environmental strains were enterotoxigenic (Burke et al., 1984). In another study, A. caviae was more often isolated from water than human clinical

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samples, and A. sobria is more often associated with clinical samples. Cytotoxin producing strains were significantly more common in patients (Millership et al., 1986). Kindschuh et al. reported that most of the clinical isolates of Aeromonas produced cytotoxin which could not be neutralized by the anti-shiga toxin antiserum. 65% of these isolates produced heat labile-like enterotoxin (LT) while only 2% of them produced enterotoxin-like activity in suckling mice assay (Kindschuh et al., 1987). The hemolytic A. veronii and related strains also caused fluid accumulation in the rabbit ileal loop test and their –hemolysin is serologically related to that of A. hydrophila (Stelma, Jr. et al., 1988). Protease of A. salmonicida, a fish pathogen, is a major virulence factor. A protease-deficient mutant was induced by mutagenesis with N-methyl-N'- nitrosoguanidine and it showed loss of virulence in determinations of LD50 doses, although it remained autoagglutinative, hemagglutinative, serum resistance, adhesive, hemolysin positive, and leukocytolysin positive (Table 5) (Sakai, 1985). Other aeromonads have been reported to be associated with human enteropathogenicity.

There are several distinct gastrointestinal syndromes commonly attributed to Aeromonas infection: acute and self-limiting watery diarrhea, vomiting, and/or fever, bloody mucoid stools, abdominal pain (Agger et al., 1985). In some geographic area, Aeromonas strains were isolated from high percentage of patients with diarrhea and dehydration. Nevertheless, Aeromonas

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infection has not been unequivocally linked to any outbreak of gastrointestinal diseases (Pazzaglia et al., 1990). In human volunteer challenges, mild-to-moderate diarrhea could be induced in only 2 of 57 human volunteers with doses of up to 5x1010 organisms of five Aeromonas strains, which had been demonstrated to produce several toxin activities (Table 6, 7) (Morgan et al., 1985).

A variety of virulence factor, such as the surface layer, capsule, different forms

of pili, toxins, quorum sensing, biofilm formation and, more recently, the type III secretion system (T3SS), were identified in various Aeromonas species that are involved in the pathogenesis.

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6.1. Invasion Recently in a Removable Intestinal Tie Adult Rabbit Diarrhea model (RITARD), Pazzaglia et al. showed that Aeromonas strains are capable of invading the mucosa of rabbit, causing diarrhea and bacteremia (Pazzaglia et al., 1990). Clinical isolates of Aeromonas were used, and in this experiment motality was 50% or greater for 7 of 12 strains; 23 of 37 rabbits that died developed diarrhea before death, and 11 of 27 surviving rabbits developed diarrhea, bacteremia was detected in 36 of 37 animal that died, but only in 2 of 27 survivors. Death, diarrhea, and bacteremia ware all strongly strain dependent (Pazzaglia et al., 1990). 6.2. Adhesion For enterotoxigenic bacteria, the ability to adhere to the intestinal mucosa is an essential early step in colonization and development of diarrhea disease. Adhesion to HEp-2 cells has been a useful model for the study of a number of pathogenic bacteria. One ml of bacteria was added to monolayer of HEp-2 cells and incubated for 90 min at 37C and washed. The adhered bacteria per cell were counted after Gram's staining. High level of adhesion was observed in 12 of 34 fecal isolates and non of the 29 environmental isolates. The proportion of high adherers (more than or equal to 10 bacteria/cell) were: A. sobria 57%, A. hydrophila 19%, A. caviae 37% (Carrello et al., 1988). Two types of pili were found: the long, thin, flexible type-L pili (mean diameter 2.5 nm and mean length 960 nm) and the shorter, thicker and straighter type-S pili (mean diameter of 5 nm and mean length of 420 nm) (Fig. 2). Type-L pili (Fig. 3) were only associated with fecal Aeromonas while type-S pili were mostly found in Aeromonas from water sources (Carrello et al., 1988). Type L pilation was associated with a high level of HEp-2 cell adhesion, and was more common in A. sobria and A. caviae than in A. hydrophila isolates. Mechanical shearing and trypsin treatment reduced the adherence of bacteria. These results suggested that adhesion may be pilus-mediated in this organism (Carrello et al., 1988).

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Pili can be observed by transmission electronmicroscopy. A drop of bacterial suspension is placed on the formvar coated grids. The grid is blotted dry with filter paper and stained with sodium silicotungstate (3%, pH 7.3) for one min, then again blotted and examined (Carrello et al., 1988). Pili can be dislodged by mechanically blending of the bacterial suspension and collected by

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centrifugation. 6.3. Hemagglutination Hemagglutination of Aeromonas isolates has been investigated. However, the role of agglutination as a virulence factor needs further clarification. A drop of bacteria suspension (in Phosphate buffer saline, PBS) is added to one drop of washed erythrocyte plus one drop of PBS to observe the hemagglutination (Crichton and Walker, 1985). According to the sensitivity of hemagglutination to the inhibition of L-fucose, D-galactose and D-mannose, the hemagglutination of Aeromonas spp. can be divided into several patterns. Crichton and Walker (Crichton and Walker, 1985) described three types of hemagglutinins in Aeromonas, (i) a mannose-sensitive hemagglutinin with strongest activity for guinea-pig red blood cells; (ii) a L-fucose or D-mannose sensitive hemagglutinin; (iii) a mannose-resistant hemagglutinin. Distribution of the hemagglutination pattern were not consistent in clinical and environmental isolates (Table 8) (Burke et al., 1984) .

6.4. Autoagglutination Autoagglutination (AA phenotype) of mesophilic aeromonads in Brain Heart Infusion broth (l7-18 h static culture at 35C) was found to be a virulence- associated marker. There were two kinds of AA+ strains: those that

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spontaneously pelleted (SP+) and those that pelleted only after boiling for 1 h (PAB+) (Fig. 4). Pelleting of bacteria was determined by measuring the absorbance at 610 nm (Fig. 5), and it was affected by pH of the medium (Fig. 6). Most of the AA+ strains were identified as either A. sobria or A. hydrophila. Of the well-documented clinical isolates of A. sobria and A. hydrophila available, 46% of strains from invasive disease and 14% of strains from noninvasive disease were SP- PAB+. The SP-PAB+ phenotype was significantly associated with invasive infection. All the SP-PAB+ A. sobria and A. hydrophila isolates killed mice within 48 h after intraperitoneal infection, and shared common O somatic antigens and possessed an external layer peripheral to the cell wall. It suggests the association of virulence to the autoagglutination and this external layer (Table 9, Fig.6) (Janda et al., 1987).

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However, Santos et al. showed that the production of siderophores, agglutination in acriflavine, and precipitation after boiling were found not to be useful tests for screening virulent strains (Santos et al., 1988). 6.5. Hydrophobicity Sixty-one isolates of Aeromonas were screened for cell-surface hydrophobicity, by determining their adhesion to a polystyrene surface (Petri dish surface) (Table 10). Isolates with type-L pilation tended to be non-hydrophobic, while type-S pilation was equally distributed between hydrophobic and non-hydrophobic isolates. The majority of the type-L pilated, non-hydrophobic isolates were highly adhesive to HEp-2 cells (Carrello et al., 1988).

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In another paper, most of the Aeromonas isolates tested were hydrophobic in several tests conducted (Table 9), it seems that hydrophobicity may not be related to the virulence of Aeromonas (Janda et al., 1987). Hydrophobicity, autoagglutination and hemagglutination are surface properties. In addition, aeromonads can be grouped according to surface properties, such as precipitation after boiling (PAB+), serotyping, resistance to lysis by bacteriophage Aehl, and possession of a surface layer (S layer) (Paula et al., 1988). 6.6. Hemolysins and Enterotoxins A number of toxins are produced by Aeromonas species; and it is now clear that both cytotoxic and cytotonic enterotoxins of Aeromonas species are capable of producing fluid accumulation in suckling mice, so that the use of such model would not allow detection of neutralization of one toxin in the presence of the other (Potomski et al., 1987a). 6.6.1. Cross-Reactive to Cholera Toxin There are two types of toxic factors produced by Aeromonas species: a cholera cross-reactive factor and non-cholera toxin cross-reactive enterotoxin (Chopra et al., 1986; James et al., 1982). Enterotoxins of other organism may also

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cross-react with cholera-toxin (CT) and heat-labile enterotoxin (LT). These include the heat-labile toxins of Klebsiella pneumonae, Enterobacter cloacae, Salmonella toxin, etc. (Honda et al., 1985). Pre-incubation with anti-cholera toxin antiserum significantly reduced intestinal secretion induced by cell-free broth preparation of A. hydrophila in rats in vivo (Fig.7). Rats immunized with cholera toxin significantly protected against enterotoxigenic A. hydrophila (Fig. 8) (James et al., 1982). Honda et al. demonstrated by ELISA with anti-cholera enterotoxin antibody that cholera toxin cross-reactive factor was produced by 5 of 14 strains of A. hydrophila and 4 or 15 strains of A. sobria, and this factor differed from hemolysin and from toxin that was active in the suckling mouse test (Honda et al., 1985).

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A cytolytic enterotoxin (detected by ELISA by using anti-cholera antiserum) was purified and characterized from a human clinical isolate by Rose et al. (Rose et al., 1989a). The toxin produced by a human diarrheal isolate of A. hydrophila was purified by ammonium sulfate precipitation, hydrophobic chromatography using phenyl-Sepharose, anion-exchange chromatography on DEAE Bio-Gel A, and size-exclusion high-performance liquid chromatography (Table 11). The factor was a single polypeptide with an apparent molecular weight of 52,000 under reducing condition.

Automated amino acid sequence analysis confirmed that the toxin was a single chain and established a 25-residue N-terminal segment which was identical to that of aerolysin (another Aeromonas toxin). However, the amino acid compositions of these two toxins differed significantly (Table 12). The composition indicated that the cytolytic enterotoxin contained few half-cystinyl residues, which explained why the molecular weight obtained from nonreducing gel conditions was virtually the same as that obtained from reducing conditions (Rose et al., 1989a).

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This toxin reacted with anti-cholera toxin antibody when tested in an ELISA assay and by immunoblot analysis. Homologous antibodies neutralized the cytotoxic and hemolytic activities associated with this toxin, but anti-cholera toxin antibody did not neutralize these activities (Table 13) (James et al., 1982; Rose et al., 1989a). This toxin also possessed enterotoxic activity as demonstrated by fluid accumulation in rabbit ligated intestinal loops (so this toxin is a so called cytolytic enterotoxin). Hemolytic activity and cholera toxin cross-reactivity of this toxin were inactivated by heat (56C 10 min) (Table 14, Fig. 9) and pH (Fig. 10). When purified toxin was injected intravenously into mice, death occurred within 2 min, whereas mice injected with whole cells or sonicated cell fragments died after several hours or days (Table 15). Results from 51Cr release experiments demonstrated that the Cytolytic enterotoxin had significant membrane- damaging capability (Rose et al., 1989b).

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Another cholera toxin cross-reactive enterotoxin was isolated by affinity chromatography (using sheep anti-cholera toxin antiserum) from crude culture filtrate of A. sobria. Major bands of the eluents were of molecular weight 43,500, 29,500, 27,000, 150,000. Treatment with 2-mercaptoethanol did not change the electrophoretic pattern of the eluted material. The rat ileal loops, infant mice and Y1 adrenal cells toxigenicities of this toxin were neutralized by cholera antiserum. There was no hemolytic or cytotoxic activity (Potomski et al., 1987a). 6.6.2. Non-cross-reactive to Cholera Toxin Two cytotoxic, cytotonic hemolysins were isolated from A. hydrophila strain AH-1 and CA-11 (Asao et al., 1986; Asao et al., 1984). These two toxins caused fluid accumulation in infant mouse intestines and rabbit intestinal loops and killed Vero cells (Fig. 11, 12) (Asao et al., 1984). Immunodiffusion test and neutralization tests with anti-hemolysin antiserum also demonstrated immunological cross-reactivity between the AH-l and CA-ll hemolysins. These two toxins had different mobility ln PAGE and electrofocusing and different amino acid composition (Table 16) (Kozaki et al., 1987). This purified toxin from AH-l is called aerolysin (or "Asao Toxin") and has a molecular weight of 52,000 (Asao et al., 1984). The "Asao toxin" a produced by 63% of A. sobria strains and

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by 93% of A. hydrophila strains (Notermans et al., 1986). Monoclonal antibodies against the CA-11 hemolysin were prepared and one of them neutralized the hemolytic and enterotoxic activities of the hemolysin. So, it is concluded that the same site on the hemolysin molecule is responsible for both hemolytic and enterotoxic activities (Kozaki et al., 1988).

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It is very likely that the aerolysin cloned by Chakraborty et al. (Chakraborty et al., 1986; Chakraborty et al., 1987) and Howard and Buckley (Howard et al., 1987) and the β-hemolysin purified by Asao et al. (AH-1 hemolysin) (Asao et al., 1984) are similar molecules. Comparison of similar A. hydrophila toxins is given in Table 17 (Rose et al., 1989b).

A non-cholera toxin cross-reactive cytotoxic enterotoxin of A. sobria was purified by affinity chromatography with monoclonal antibodies (Potomski et al., 1987b). The mol.wt. of this toxin is 63,000; with pI of 6.2; deactivated at 56C for

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10 min. The purified enterotoxin caused fluid accumulation in rat ileal loops and infant mice, and it was cytotoxic to cultured cells, hemolytic to human erythrocytes, and lethal to mice after intravenous injection. This toxin did not cross react with cholera toxin and its activity was not neutralized by antiserum to cholera toxin (Fig. 13)(Potomski et al., 1987b). This toxin cross reacted with monoclonal antibody of the hemolysin from A. hydrophila CA-ll (Kozaki et al., 1989).

The hemolysin (cytotoxic enterotoxin) produced by A. sobria in minimal-salt medium containing nitrilotriacetic acid (NTA, protease inhibitor), no or little hemolysin was detected without trypsinization of all the cultures, while after trypsinization, hemolysin was detected in the same quantity as in BHI culture (Table 18). NTA has an inhibitory effect on proteolytic activity, so it may also inhibit the A. sobria protease(s) associated with activation of the hemolysin. Besides trypsin, A. sobria precursor was activated by either lysine- or arginine-specific protease (Fig. 14). This indicates that activation results from cleavage of an alterative site on the hemolysin molecule. This activation is accompanied by some reduction in the molecular size (Kozaki et al., 1989). The molecular weight of hemolysin from A. sobria before and after trypsinization were 53,000 and 49,000, respectively; and those from A. hydrophila 54,000 and 51,000, respectively (Kozaki et al., 1989).

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In immunoblotting analyses with the culture supernatant of A. sobria the

monoclonal antibody reacting specifically to A. hydrophila CA-11 hemolysin

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bound to this toxin. However, the monoclonal antibody reacting to A. hydrophila AH-l hemolysin did not react with this toxin. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin (Table 19, Fig. 15) (Kozaki et al., 1989).

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The aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus (Howard and Buckley, 1985b). A large protein of at least 250 times less hemolytic than aerolysin was isolated. This large protein and the aerolysin both had the same amino acid sequence at the amino terminus. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin (Howard and Buckley, 1985a). 6.6.3. Heat-stable enterotoxins Nine of 12 clinical isolates of A. hydrophila hybridized with ollqonucleotide probes (sequences that are not shared by E. coli LT) for cholera toxin (Table 20) (Schultz and McCardell, 1988). They also demonstrated the presence of at least two types of toxins, one is deactivated by heating at 55C for 20 min, while other type is stable by this heat treatment (Table 21). Concentrated cell-free supernatants or lysates from either culture, heated at 56C for 20 min, produced cytotonic effects in Y1 mouse adrenal cells and CHO cells and caused a 1.5- to 2.2-fold increase in production of cyclic AMP in CHO cells (Table 22). Three proteins with molecular weights of 89,900, 37,000, and 11,000 reacted with cholera-toxin antiserum (Schultz and McCardell, 1988). The 11,000 daltons band is probably the B subunit of the toxin. The 89,900 hand may be the holotoxin which has not broken down to subunits. The 37,000 band may be an A subunit that is slightly larger than either the whole A (28,000) or A1 (21,000) subunit of cholera-toxin.

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Chakrahorty et al. also demonstrated the presence of the relatively heat-stable toxin which was cytotonic to culture cells (Chakraborty et al., 1984). 6.7. Activities of Aeromonas Toxins Sensitivity of various erythrocyte species was determined and mouse is the most sensitive one and followed by monkey, guinea pig, rabbit, horse, ox, human, goat and sheep, and the activity is temperature dependent. In another experiment, erythrocytes of sheep, cow and horse were most sensitive to the hemolysins of A. hydrophila (Table 23) (Brenden and Janda, 1989).

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The 86Rb release from rabbit erythrocytes with AH-1 and CA-11 hemolysins occurred within a few minutes, preceding hemoglobin release. It suggested that both hemolysins generate transmembrane pores which permit rapid passage of cations. Since lysis of rabbit erythrocytes with each hemolysin was inhibited by Dextran 4 as an osmotic protectant, the pores generated on erythrocyte membrane may be of about 3-nm diameter (Kozaki et al., 1987). Immunoblotting analysis showed that hemolysins associated with lysed rabbit erythrocyte membrane formed oligomers with Mol. Wt. of 355,000 and 365,000, being likely to be heptamers or octamers (Fig. 16). It may be considered that such oligomers resulted solely from aggregation of hemolysin but not from association of hemolysin with some erythrocyte-membrane component, since prolonged aging or concentration of the hemolysins also induced the same oligomers (Kozaki et al., 1987). Such aggregation also occurs in staphylococcal -toxin and streptolysin O.

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During the action of cytolytic toxin, glycoprotein liberated from rat erythrocytes by trypsin treatment may be the receptor of aerolysin. In case of rabbit erythrocytes contain little or no such glycoprotein and that hemolysis of rabbit erythrocytes with aerolysin may be due to its direct interaction with the lipid bilayers. In fact, these hemolysins bound firmly to phosphoglycerides with net negative charge but weakly to the ones with no net electric charge at neutrality as analyzed by TLC immunostaining. Neither cerebrosides, gangliosids GM1, sphingomyelin nor lysophosphoglyceride reacted with the hemolysin. So, it is speculated that A. hydrophila hemolysins bind to membrane components (probably phospholipid) of rabbit erythrocytes with net negative charge, enter the membrane, and interact with the nonpolar tails of phospholipids by hydrophobic-hydrophobic interaction, eventually forming functional channels and disrupting membrane permeability. Such binding is further proved by the inhibition of sodium salicylate on the activity of both hemolysins (Fig. 17), since sodium salicylate is considered to interact with the phospholipids bilayer and to alter the membrane potential (Kozaki et al., 1987).

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Like the alpha-toxin from Staphylococcus aureus, aerolysin formed SDS-stable oligomers with apparent molecular masses of about 220 kD in n-decane. When aerolysin was added in small quantities to the aqueous solution bathing a lipid bilayer membrane, the specific conductance of the membrane increased by several orders of magnitude (Fig. 18) (Chakraborty et al., 1990).

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The results suggested that aerolysin formed an ion-permeable channels which had a single-channel conductance of about 70 pS in 0.1M KCl. The aerolysin channel appears to be smaller than the pores in the bacterial outer membranes and probably has a diameter between 0.8 and 1 nm (Chakraborty et al., 1990) instead of 3 nm estimated by the osmotic protection of Dextran 4 (Kozaki et al., 1987). A similar diameter may be estimated for the channel formed by -toxin of S. aureus (Table 24) (Chakraborty et al., 1990).

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6.8. DNA adenine methyltransferase

Among the various virulence factors produced by A. hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. DNA adenine methyltransferase gene of A. hydrophila was cloned in a T7 promoter-based vector system using E. coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila (Erova et al., 2006).

6.9. Quorum sensing

A model system using gnotobiotically cultured shrimp Artemia franciscana was developed to determine the impact of mutations in the quorum sensing systems of A. hydrophila, V. anguillarum and V. harveyi on their virulence. Mutations in the autoinducer 2 (AI-2) synthase gene luxS, the AI-2 receptor gene luxP or the response regulator gene luxO of the dual channel quorum sensing system of V. harveyi abolished virulence of the strain towards Artemia. Moreover,

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the addition of an exogenous source of AI-2 could restore the virulence of an AI-2 non-producing mutant. In contrast, none of the mutations in either the acylated homoserine lactone (AHL)-mediated component of the V. harveyi system or the quorum sensing systems of A. hydrophila and V. anguillarum had an impact on virulence of these bacteria towards Artemia. This results indicate that disruption of quorum sensing could be a good alternative strategy to combat infections caused by V. harvey (Defoirdt et al., 2005).

6.10. Type III secretion system

The gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila was deleted, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme (Fig. ), and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses (Fig. ). In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. The production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production (Sha et al., 2005).

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6.11. Alternative host model

The use of unicellular amoebae allows a very simple assessment of bacterial virulence in many different pathogens. In a typical experiment, Dictyostelium cells form a phagocytosis plaque on a lawn of nonpathogenic bacteria but not on a lawn of pathogenic bacteria (Fig. ). The virulence of bacteria can thus be extrapolated from their ability to sustain Dictyostelium growth, as shown previously for Klebsiella pneumoniae or Pseudomonas aeruginosa (Froquet et al., 2007).

7. CONCLUSION Aeromonas spp. may be important foodborne pathogens in the future. A large proportion of isolates from food and environment produce hemolysin and other

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toxic factors. Some of these factors may be involved in the enterpathogenicity of these bacteria. A number of studies showed the presence of various kinds of toxins, however, the properties of these toxins were sometimes contradictory. It may be due to: (a) presence of numerous different toxins in different strains of Aeromonas spp., (b) variation of toxins occurs frequently, (c) presence of co-factors which affect the properties of toxins. Also, a number of reports showed the presence of cholera-toxin cross-reacting toxins, but only oligonucleotides of the cholera-toxin hybridized with the Aeromonas DNA. Variation may occur during evolution. Aeromonas widely distributes in the food and environment, the following topics must be investigated in the future to clarify the role of Aeromonas in food poisoning: (a) Virulent factor involved in enteropathogenicity. (b) Incidence and enteropathogenicity of Aeromonas spp. in different foods. (c) The path of distribution of pathogenic Aeromonas in the nature. Also, a rapid and accurate method for the isolation and identification of pathogenic Aeromonas is required for detail survey. 8. REFERENCES

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