Aeromonas hydrophila and related species are Gram negative, aerobic and facultative anaerobic freshwater bacteria, which is widely distributed in the aquatic environment. It has been related to disease problems in humans and are also pathogenic for aquatic and terrestrial animals (Cascon et al., 1996). It has been responsible for haemorrhagic septicaemia or motile Aeromonas septicaemia, infectious dropsy, red mouth disease, red pest disease in fish and reported to be associated with Epizootic ulcerative syndrome (EUS) in South East Asian countries and India (Roberts et al., 1992). Most environmental isolates of A.hydrophila and related aeromonads secrete many extracellular products, some of which, such as aerolysin, haemolysin, enterotoxin, proteases and cytolytic enterotoxin are considered as virulence factors in pathogenesis (Cascon et al., 1996; Sirirat, et al., 1999). Since A.hydrophila was first recognized a an important pathogen for aquatic animals and significant opportunistic pathogen for humans having public health significance, many efforts were dedicated to find methods for accurate identification and classification of species belonging to this genus (Cascon et al., 1996).
Aeromonas spp. isolates are known to be phenotypically, serologically and genetically quite diverse and the conventional methods of identifying these microorganisms like microbiological culture, biochemical tests, protein analysis, serotyping etc. give contradictory results. Biochemical tests are known to be time consuming and sometimes lead to confusion in diagnosis of species of pathogens. Again serological tests are sometimes handicapped by lack of specificity of antisera and cross reactivity. It appears that a rapid and accurate identification of the genospecies of Aeromonas would be a useful diagnostic tool in clinical and veterinary laboratories. Attempts have been made by application of molecular techniques for prompt identification, classification and characterization of pathogens. Different molecular genomic methods like polymerase chain reaction (PCR), Randomly amplified polymorphic DNA (RAPD), Restriction fragment length polymorphism (RFLP), Ribotyping etc. are now commonly used to identify and characterize this pathogen. RAPD-PCR, as developed by Williams et al., (1990) and Welsh and McClelland (1990), has been used to characterize populations and strains of animals, plants , fungi and bacteria (Hardeys et al., 1992; Lawrence et al., 1993) and has been proved useful for identification and classification of a variety of microbial pathogens of humans and animals (Berg et al., 1994). In the present study Aeromonas spp. obtained from diseased fish were screened using biochemical tests, PCR and and characterized using RAPD-PCR to study their genomic variability.
Materials and Methods
Processing of sample and microbiological analysis
Water and sediment samples were collected from different fish farms, beels and bheries located in West Bengal (Table 1), were brought to the laboratory under iced condition for analysis. Samples were plated onto Tryptic soya broth and agar (Difco) and Aeromonas selective growth medium (Rimler-shotts, RS Medium, HiMedia Mumbai). The plates were incubated at 30oC for 24 hrs and observed for characteristic colony development. The bacterial colonies were then identified using a battery of standard cultural and biochemical tests, as per Burgey’s manual of determinative bacteriology (Buchanan and Gibbons., 1988).
Phenotype characterization :
Selected bacterial isolates were also tested using Biolog -Microlog 4.2, automated microbial identification system (Biolog Inc, Hayward, CA, USA), which used to measure utilization of 95 separate carbon sources following methods as recommended by the manufacturer. The Biolog system consisted of a microplate containing 95 different carbon sources and a control well, a turbiditymeter and a computer-driven automatic plate reader. The Biolog plates can be read at 4 or 24 hr. For the present study, we chose to read plates only at the 24hr period. Standard reference strain of A.hydrophila, A.sobria, obtained from MTCC, Chandigarh were used, along with other isolates.
Extraction of bacterial DNA and Plasmids
Bacterial DNA from above Aeromonas isolates were extracted from broth cultures by treatment of cell-pellet with Lysozyme, 10% SDS and Proteinase K followed by extraction with Phenol – chloroform and precipitation with 5M NaCl and 2 volume of ethanol (Sambrook et al., 1989). The DNA strands thus obtained, were separated by centrifugation and dissolved in TE buffer. Bacterial plasmids were isolated from broth cultures using Alkaline lysis method (Sambrook et al., 1989), by treatment of culture pellet with 0.2 N NaOH, 1% SDS, 3M Pot. acetate followed by extraction with Phenol-chloroform and precipitation with 2 volume of chilled ethanol. The plasmids were analysed on 1% agarose gel and visualized using Gel documentation system (GelDoc2000, Biorad).
Detection of Aeromonas isolates using PCR
PCR was standardized and used for specific detection of A.hydrophila DNA group1, Aerolysin positive A. hydrophila and virulent (enterotoxigenic, hemolysin) Aeromonas spp. from samples of fish disease outbreaks and microbial samples. Primers specific for hemolysin gene (232 bp product, Kingombe et al., 1999) and ‘aer’ gene (209 bp product, Ozbas et al., 2000) were used as the target genes for PCR amplification. A PCR mix of 50 µl containing Taq polymerase enzyme, 10X buffer, primer 1 & 2, dNTP mix and bacterial sample lysate was kept in PCR tube and amplified using Thermalcycler, Gene Amp2400 system. After 30 cycles of amplification (at 94o C 1 min, 55oC 1 min and 72o C 1 min), the products were electrophoresed on 1% agarose gel and visualized under Gel documentation system. Following primers were used :
AH CF1 : 5’ –GAG-AAG-CTC-ACC-ACC-AAG-AAC-A-3’
AHCR2 :5’- AAC-TGA-CAT-CGG-CCT-TGA-ACT-C-3’
AER1: 5’- CCA-AGG-GGT-CTG-TGG-CGA-CA-3’
AER2: 5’- TTT-CAC-CGG-TAA-CAG-GAT-TG-3’
RAPD-PCR analysis of genomic DNA and plasmid
The selected Aeromonas isolates of after cultural and biochemical tests, were used in RAPD-PCR analysis, using the standard RAPD method on the line of Miyata et al., (1995). The extracted genomic DNA and plasmids were amplified separately. Before RAPD amplification of samples, one sample DNA and plasmid were screened using a set of 20 oligo primers (OPA1- OPA10, AH1-AH10), to see the suitability of primers in producing polymorphic bands. The amplification was carried out using Thermal cycler (Gene Amp PCR 2400 system, Biosystems, USA) using the programme as follows : One cycle of initial denaturation at 94oC x 4 min. followed by 45 cycles of 94oC for 45 sec., 36oC x 45 sec., and 72oC x 90 sec. Final extension of 72oC x 7min was given and the product was stored at 4oC till analysed. The PCR amplified products were analysed on 1.5% agarose gel following the standard protocol (Sambrook et al., 1989). Accordingly 5 primers, which give polymorphic bands were selected and further used in RAPD-PCR analysis of all Aeromonas genomic DNA and plasmid samples. PCR amplification was carried out using the same amplification cycle as above and electrophoresed on 1.5% agarose gel. The banding pattern and RAPD profiles were analysed using the Gel documentation system and Quantity-1software (Geldoc 2000, BioRad). Dendogram, phylogenic analysis and similarity index were calculated using Ntsys and TFPGA softwares. The similarity index between the isolates were estimated as per Nei and Li (Dice) method.
Results
Isolation of pathogens and infection trials
Water and sediment samples were collected from different fish farms/Beels and Bheries (Table 1) were tested using microbiological techniques. Samples were inoculated to general and selective bacteriological media. Cultural and biochemical tests were also carried out and the results have been presented in Table 2. Samples were also analyzed using BIOLOG Automated Bacteriological identification system. Different groups of bacteria belonging to different Aeromonas gropus viz. Aeromonas hydrophila DNA gp.1&2 (15nos), A.veronii b.v. sobria DNA gp.8(24nos), A.sobria (5 nos), A.enchelia(4nos), A.icthiosmia (4 nos), A.veronii DNAgp.10(2 nos) and unidentified Aeromonas spp. (28 nos) (Table 1). However there were also some Pseudomonas spp. identified. Only Aeromonas spp. were further characterized using PCR and RAPD-PCR.
Detection of Aeromonas isolates using PCR
PCR was standardized and used for specific detection of A.hydrophila DNA group1, Aerolysin positive A. hydrophila and virulent (enterotoxigenic, hemolysin) Aeromonas spp. from samples of fish disease outbreaks and microbial samples. Primers specific for hemolysin gene (232 bp product) and ‘aer’ gene (209 bp product) were used as the target genes for PCR amplification. Encouraging results were found using screening test and comparing known positive samples. All A. hydrophila as identified using Biolog system gave amplification of 209bp DNA band indicating them to be aerolysin gene positive. All Aeromonas samples gave amplification of 232 bp DNA indicating these to be hemolysin gene positive.
RAPD-PCR analysis of Aeromonas isolates
RAPD based on PCR amplification using random primers, was employed to detect and study genomic variability in A.hydrophila isolates collected form cases of disease in fish and shellfish. A set of 20 random primers (AH1 to AH10 and OPA 1- OPA 10) were employed using one A.hydrophila isolate to test the suitability of each primer. After electrophoresis on 1% agarose gel, variable banding pattern were visualized with each primer and band numbers ranged between 3 and 8. To avoid non-reproducible results and non-specific bands, only clearly visible bands were taken into account and faint bands in gel were avoided. From this, optimum banding pattern were obtained with AH1, AH3, AH8, OPA5 and OPA 9 primers. Hence these five primers were selected for further screening of all Aeromonas isolates in RAPD-PCR. The common bands, similar bands and unique bands were marked and their molecular weight were estimated using the gel documentation system. The genetic similarity index was calculated as per the formula of Nei and Li using Bio1D and TFPGA softwares. Dendogramic analysis of all 70 isolates revealed high genetic variability among the isolates regardless of the fish species or organs used for bacterial isolation (Fig.1). Out of 70 isolates analyzed, 4 major distinct groups could be revealed and the similarity ranged between 0% and 65%.. There was high degree of similarity among A.vernii b.v. sobria, A.enchelia, A.icthismia with nearly 95% similarity. However the similarity of A.veroniib.v. sobria with A.hydrophila was around 85%. The unidentified Aeromonas isolates for 6 sub-groups with around 90% similarity among them and 65% similarity with other identified Aeromonas species.
Discussion
In the present study, water and sediment samples were collected from different fish farms, beels, bheries were screened using microbiological techniques. Single colonies were isolated and pure cultures were then tested using a battery of biochemical tests and also using BiologGN plates. A total number of 68 Aeromonas species belonging to different groups like A.hydrophila DNA group 1; A.hydrophila like DNA group-2 A. veronii b.v. sorbia DNA group8; A.sobria ; A. enchelia ; A. icthiosmia; and 28 unidentified Aeromonas spp. were identified. Biolog system of analysis has also been used by a number of investigators. Where as Miller et al.,(1993) observed overall accuracies (correct to the species level) upon initial testing between 69% and 74%, Klingler et al., (1992) reported 98% accuracy upto genus level and 76% to species level. Nielson et al., (2001) in their investigation of moribund fish and crabs in Xhejiang province of China, could isolate 88 bacterial isolates. Out of 69 motile aeromonads, 35 were identified as A.hydrophila and other motile aeromonas spp. were also isolated, In the present study we found the Biolog system easy to use, to update and to customize for specific needs. Its accuracy in identifying Aeromonas spp. was satisfactory. Again the results biochemical tests supported biochemical fingerprinting results using Biolog system. However, incorrect identification of other microbial isolates and identification after variable incubation time detract the usefulness and applicability of this system as also reported by others (Klingler et al., 1992).
These bacteria were subsequently screened using PCR and characterized using RAPD. RAPD technique as developed in 1990 by Williams et al., have been used to demonstrate simple and reproducible DNA fingerprinting. Randomly amplified DNA fragments of genomic DNA are obtained from PCR using randomly designed short primers. Both genomic variation between bacterial species and genetic polymorphism between bacterial strains could be identified as differences in the sizes and numbers of DNA fragments obtained. In the present study, a phylogenetic analysis of Aeromonas spp. was carried out using RAPD-PCR. Out of 10 primers screened to test their suitability to produce multiple, polymorphic bands, 5 primers were selected and used for RAPD-PCR With each primer and each isolate, DNA bands of variable size and intensity could be visualized on agarose gel electrophoresis. After scanning the gel using Gel Documentation System, DNA bands could be observed. The common bands, similar bands and unique bands were marked and their molecular weight were estimated using the gel documentation system. The genetic similarity index was calculated as per the formula of Nei and Li using TFPGA software. Dendogramic analysis of all isolates revealed high genetic variability among the isolates regardless of the fish species or organs used for bacterial isolation. Out of 70 isolates analyzed, 4 distinct groups could be revealed and the similarity ranged between 0% and 65%.. There was high degree of similarity among A.vernii b.v. sobria, A.enchelia, A.icthismia with nearly 95% similarity. However the similarity of A.veroniib.v. sobria with A.hydrophila was around 85%. The unidentified Aeromonas isolates for 6 sub-groups with around 90% similarity among them and 65% similarity with other identified Aeromonas species.
In the present study we observed RAPD profiles to be highly reproducible when only clear bands were taken into account, avoiding faint and low molecular weight bands. Similar observations were also made by Miyata et al., (1995), who reported that occurrence of faint products were not caused from genomic differentiation but depended on purity of template DNA. They also obsrrved that the sizes of both the clear and faint products of A.salmonicida subsp. salmonicida were identical. The contaminated small molecular weight nucleic acids may form the template-primer complexes which decrease the frequency of PCR amplification (Miyata et al., 1995). RAPD-PCR has been successfully used to characterize several species of bacteria. Sudeesh et al., (2002) used RAPD fingerprinting to study the genomic diversity within V.parahaemolyticus and V.alginolyticus strains isolated from cultured shrimps and revealed conglomeration of 15 strains of V.parahaemolyticus and 9 strains of V.alginolyticus into two distinct groups. Distinct clusters were also observed within each species indicating genomic diversity in these bacteria. Similar was also the observation in the present study. A.hydrophila isolates revealed high genetic diversity regardless of the fish species or organs used for bacterial isolation. Out of 42 isolates analyzed, 9 distinct groups could be revealed and the similarity ranged between 0% and 65%.. There was high degree of similarity among A.vernii b.v. sobria, A.enchelia, A.icthismia with nearly 95% similarity. However the similarity of A.veroniib.v. sobria with A.hydrophila was around 87%. The unidentified Aeromonas isolates for 4 sub-groups with around 90% similarity among them and 65% similarity with other identified Aeromonas species. This indicated high genomic variability of Aeromonas isolates.
S.S. Mishra, Mitali Dhiman, B.K. Behera and M.K.Das