Tomato Collection: Commercial tomatoes were purchased from Ananda market, Mirpur market and Jatrabari market of Dhaka city, Bangladesh and were processed within 24 h of collection. Two kilograms of tomatoes from each market were collected in sterilized ziploc bag, and transported to the laboratory in a cool box and were processed within 24 h of collection. Each tomato weighed 40.0 ± 0.5 g as measured by an electric balance (Shimadzu Corporation, Tokyo, Japan). Injured or dirty tomatoes were discarded.
Washing Solution and Sample: Washing ASC and chlorine water were used as washing solutions and were prepared immediately prior to application. The final concentration of acidified sodium chlorite solutions was adjusted to 0.5 g/L of sodium chlorite and 1 g/L of citric acid. The chlorine solution was prepared using sodium hypochlorite (Wako Chemical Co. Ltd, Osaka, Japan) solution to distilled water (vol/vol), and concentration was adjusted to 200 ppm. Distilled water (DW) was used as a control. In each experimental condition, one tomato (ca. 40 g) was washed with 250 mL of the washing solution in a sterile beaker (1 L). Washing was carried out for 2 min at room temperature with gentle agitation by using a glass rod. After washing, the solution was decanted, and tomatoes were rinsed with 250 mL of distilled water. Then the tomatoes were placed on a sterile perforated tray to drain off the excessive water and placed in laminar flow bio-safety cabinet to facilitate drying for 2 hours.
Sample Processing and Microbiological Analysis: After washing and drying, the tomatoes were aseptically cut into small pieces and ten grams of sliced tomato were placed in a stomacher bag with 90 mL of sterile saline water. Diluted and undiluted samples were surface plated on both selective and nonselective agar medium. Tryptic soy agar (TSA; Oxoid) was used as a nonselective medium for determination of viable cells number. Coliform agar; sorbitol macconkey agar (SMAC) supplemented with cefixime (0.05 mg/liter) and potassium tellurite (2.5 mg/liter) (CT-selective supplement, Oxoid); bismuth sulfite agar (BSA; Oxoid); listeria selective agar supplemented with SR0227E and yersinia selective agar supplemented with yersinia selective supplement were used as selective medium for the determination of coliform bacteria, E. coli O157:H7, Salmonella spp., L. monocytogens and Y. enterocolitica respectively. After incubation of enumeration media at 37°C for 24 to 48 h at least five presumptive colonies of Salmonella, E. coli O157:H7 and L. monocytogenes were confirmed with direct immunoassay test kit (Universal Health Watch, Columbia, MD, USA.). All experiments were repeated five times to confirm reproducibility.
Antibiotic Susceptibility of Isolated Pathogens from Tomato: Susceptibility of isolated bacteria to different antimicrobial agents was determined in vitro according to Bauer-Kirby disc diffusion methods. Commercially available antimicrobial discs (Oxoid Ltd., Basing stoke, Hants, UK) of ampicilin (AMP = 25 μg), amoxicillin (AML = 10 μg), azithromycin (AZM = 15 μg), amikacin (AK = 30 μg), aztronem (ATM = 30 μg), cefrixone (CRO = 30 μg), cefotaxime (CTX = 30 μg), cefuroxime (CXM = 30 μg), chloramphenicol (C = 30 μg), cefepime (FEP = 30 μg), cephalexin (CL = 30 μg), clindamycin (DA = 12 μg), doxycycline (DO = 30 μg), erythromycin (E = 50 μg), gentamycin (CN = 10 μg), kanamycin (K = 30 μg), levofloxacine (LEV = 5 μg), nitrofuran (F = 300 μg), oxytetracycline (OT = 30 μg), rifampicin (RD = 5 μg), oxacillin (OX = 1 μg), vancomycin (VA = 30 μg), imipenem (IPM = 10 μg) and sulphamethoxozole (SXT = 25μg) were used to detect the zone of inhibition around the antibiotic disc.
Plasmid DNA Extraction: Plasmid DNA was extracted from the fresh tomato isolates grown in Luria Broth (LB) medium and incubated at 37°C overnight with mild shaking (180Xg). After incubation, 1 mL of the culture was taken and centrifuged (16,000 X g; 30 s) at 4°C. The supernatant was removed and the pellet was resuspended in 150 μL of Tris-EDTA buffer 10 mM Tris chloride (pH 8), 1 mM EDTA (pH 8) solution by vigorous vortexing. Two hundred microliter of NaOH-SDS (0.2 M NaOH, 1% SDS) solution and 150 μL of 3 M potassium acetate (pH 4.8) were then added and vortexed for 10 s. The content was centrifuged (16,000 X g; 5 min) again at 4°C and the supernatant was precipitated with 600 μL of ice cold ethanol. A portion (15 μL) of plasmid DNA was loaded on to a 1.0% agarose gel containing 0.5 μg mL-1 ethidium bromide and electrophoresed in TBE (Tris-Boric acid-EDTA) buffer. The plasmid DNA were visualized by placing the gel on a UV (300 nm) transilluminator and recorded using the digital documentation imaging system (model universal Hood II; BIO-RAD Laboratories, Hercules, CA, USA).
Statistical Analysis: All trials were replicated five times. Reported plate count data represent the mean values obtained from five individual trials, with each of these values being obtained from duplicated samples. Data were subjected to analysis of variance using the Microsoft Excel program (Redmond, Washington DC, USA.). Significant differences in plate count data were established by the least-significant difference at the 5% level of significance.