Study on Outer Membrane Protein (OMP) Profile of Aeromonas Strains using SDS-PAGE

Mesophilic aeromonads are being increasingly reported pathogen of humans and lower vertebrates. Water and foods are considered to be the chief source of Aeromonas spp. At present there are several techniques available for the detection of Aeromonas spp. from water and foods. However, there is still need to develop immunodiagnostics for rapid detection of Aeromonas spp. irrespective of their species or serotype. To meet out this requirement present study was undertaken to identify the common protein moiety in their OMPs by SDSPAGE so, that immunoassays can be developed for efficient and rapid detection of Aeromonas spp. from foods.


Introduction
Chimnitz and named Bacillus punctatus (Zimmerman, 1890).A variety of foods and food Emergence of Aeromonas spp. as an important products including seafood, chicken and red human pathogen has led to a considerable interest meat, vegetables, raw milk and its products have in the organism in last two decades.Aeromonads been shown to harbour Aeromonas spp.( (BSA) were prepared in distilled water to a total Aeromonas strains were isolated from volume of 400 µl.To all the tubes, 400 µl of 2x different sources such as chicken meat, fish, milk, lowry concentrate was added and incubated at room temperature for a minimum of 10 min.egg etc. using Buffered Cephalothin Dextrin Thereafter, 200 µl of the 0.2 N Folin reagents was Broth -10 (BCDB-10) as selective enrichment o added and incubated (55 C for 5 min).The broth.Isolates were serotyped by the courtesy of Absorbances were read at 650 nm using Dr. T. Shimada, Chief, Laboratory of Enteric polystyrene cuvettes.A standard graph was Infection 1, National Institute of Health, Tokyo, plotted against optical density (OD) of BSA in 162, Japan (Table 1).different concentrations.Protein concentration of finally, destained with destaining solution.OMP of 16 Aeromonas strains was separated as Calculation of molecular weight(s) (MW) of the per the method of Crosa and Hodges (1981) as peptide(s) was done by extrapolation of relative modified by Santos et al. (1996).Briefly, mobility of the unknown samples against that of Aeromonas grown overnight in 100 ml of tryptic standard molecular weight markers.Details of soya broth (TSB, Difco) were recovered by reagents used for SDS-PAGE are given in table-2.centrifugation at 5000 rpm for 30 min.Cells were resuspended in 3 ml of 10 m mol/l tris buffer Results and Discussion containing 0.3% (w/v) (pH 8.0) and sonicated The separation of OMP was carried out by with a sonifier (MSE, Ultrasonicator) (10 amplitude, culturing Aeromonas strains in brain heart 45 seconds, 4-5 times).After centrifugation at 0 infusion broth at 37 C with shaking, followed by 10,000 g for 2 min.the supernatant fluids were ultrasonification and centrifugation.Inner and transferred to new tubes and centrifuged for 1 hr 0 outer membranes were separated by incubation at 17,000 g at 4 C. Cell envelop suspensions were with sarkosyl.This separation was necessary incubated with 3% sodium lauroyl sarcosinate because OMP profiles are reported to be (sarcosyl) (w/v) in 10 m mol/l tris buffer at room influenced by temperature and air supply to temperature for 20 min.Outer membrane protein bacterial cultures (Statner et al., 1988).This was obtained by centrifugation at 17000 g for 1 hr method was able to recover the whole OMPs and washed twice with distilled water.The OMP from Aeromonas strains (Kuijper et al., 1989).
o was stored at -20 C.
OMP of 16 strains were separated and protein Protein estimation: The protein content was concentration estimated is presented in table 2. In determined by the method of Schaterle and this study protein concentrations were in the Pollack (1973) with little modifications.A small range of 11.3 mg/ml to 16.2 mg/ml.

SDS-PAGE analysis of
OMPs: SDS-PAGE few minor bands in the lower molecular weight analysis of OMP of different Aeromonas strains range (10-12 kDa) were also observed in certain revealed up to 4 major and 4 to 6 minor strains.polypeptide bands (Fig.1& 2).The molecular Major polypeptides of 14 and 35 kDa and weight (MW) of the major polypeptide bands, minor polypeptide 25 kDa were common in most estimated by comparison with standard MW of the Aeromonas spp.irrespective of serogroup markers run parallel was in the range of 14 to or species.Heterogeneity in protein profile was 55kDa.Large smearing of the bands in the MW observed among Aeromonas strains of different range of 40 to 45 kDa was observed.In addition a serogroups as well as of same serogroups.
The first isolation of wound infections, wounds exposed to water are Aeromonas was made in 1890 from tap water in likely to be infected with Aeromonas spp.amount (25 µl) of OMP was diluted with 400 µl of (Skiendzielewski and O'Keefe, 1990).distilled water.Different concentrations (25, 50, 75, 100, and 125 µg) of bovine serum albumin Hunter are ubiquitous in nature being isolated from wide and Burke, 1987; Majeed et al., 1989; Palumbo et variety of sources.They are normal flora of al.,1989; Kirov et al., 1990).Cases of Aeromonas aquatic and terrestrial animals as well as etiologic associated gastroenteritis have been reported agents of disease in numerous cold-blooded and from all over the world including Australia warm blooded animals (Cahill 1990; Joseph and (Burke et al., 1983), Bangladesh (Sack et al., Carnahan, 1994).The aquatic environment is 1987), Brazil (Schorling et al., 1990), Ethiopia considered to be the principal reservoir of (Wadstrom et al., 1976).