Molecular Diversity Analysis of Aeromonas species isolated from fish in West Bengal By Mitali Dhiman and S. S. Mishra

Bacteria belonging to the genus Aeromonas, family Aeromonadaceae

, are widespread in the environment, especially freshwater brackish water, estuarine environment and in sewage. These are also involved in a wide spectrum of diseases of human infections and animals, including fish, frogs, reptiles, birds and cattle. Aeromonas have 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). Among Aeromonas species, A.hydrophila, A caviae, A.veronii, A.eucrenophila, A. popoffii and A.culicola are predominating species isolated from human cases where as A. hydrophila, A sobria, A veronii b.v. sobria have been found fish samples. Aeromonas spp. 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. Alternative specific genomic fingerprints have been proposed as diagnostic tools by means of amplification of interspersed repetitive DNA sequences present in bacterial genomes or by amplification of random sequences by arbitrary primers, RAPD (Williams et al., 1990).

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, 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).

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. 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

Different groups of bacteria belonging to different Aeromonas gropus viz. Aeromonas hydrophila DNA gp.1&2, A.veronii b.v. sobria DNA gp.8, A.sobria, A.enchelia, A.icthiosmia), A.veronii DNAgp.10 and unidentified Aeromonas spp.were isolated and identified. 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.

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 isolates revealed high genetic variability among the isolates regardless of the fish species or organs used for bacterial isolation. Out of these 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.

Molecular Diversity Analysis of Aeromonas species isolated from fish in West Bengal By Mitali Dhiman and S. S. Mishra

By: MITALI DHIMAN

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