Study area, sampling and sample processing. Total 50 samples of Brassica oleracea (25 were grown with bio-fertilizer and 25 were grown using chemical fertilizer) were randomly collected from different rural area in Bangladesh during January 2014-March 2014 following standard protocol.
Estimation of total viable bacteria and fungi. The enumeration was performed by using 0.1 ml of each sample from the dilution 10-3 was spread onto nutrient agar (NA) and Sabouraud Dextrose Agar (SDA) for total viable bacteria (TVB) and total fungal load, respectively. After that the nutrient agar (NA) and Sabouraud Dextrose Agar (SDA) plates were incubated at 37oC for 24 hours and at 25oC for 48 hours for the detection of total viable bacteria (TVB) and total fungus respectively according to the standard guideline.
Estimation of coliform count and fecal coliform count (FCC). An aliquot of 0.1 ml of each sample was spread on to MacConkey agar, and membrane fecal coliform agar plates for the estimation of coliform (E. coli and Klebsiella spp.) and fecal coliform (FCC) respectively. For coliform count, all plates were incubated at 37°C for 24 hours while for estimating the fecal coliforms, incubation was carried out at 44.5°C for 24 hours. Eosin methylene blue agar media was further used for the observation of production of green metallic sheen (if any) to ensure the specific characteristic of E. coli strains.
Estimation of Staphylococcus spp., Pseudomonas spp. Clostridium spp. Bacillus spp. and Listeria spp. Same amount of samples as described above was spread on to mannitol salt agar, cetrimide agar Starch agar and Listeria media for the isolation of S. aureus, Pseudomonas spp., Bacillus spp and Listeria spp., respectively. Afterwards, plates were incubated at 37°C for 24 hours. For the isolation of clostridium 1 ml of each blended sample were mixed in sterile normal saline in a ratio of 1:8 followed by heating at 80°C for 15 minutes in order to kill vegetative cells of the microorganisms (17,18). Furthermore, 1ml of each samples were introduced into 9 ml of fluid thioglycolate broth and incubated for 4 hours at 37°C. Then 0.1 ml of each sample from this enriched broth was poured on Clostridium isolation agar plates according to pour plate method and were incubated at 37°C in anaerobic condition for 48 hours.
Estimation of Salmonella spp., Shigella spp. and Vibrio spp. For the isolation of VBNC Salmonella spp., Shigella spp., and Vibrio spp. 1ml of each samples was inoculated into Alkaline peptone water (APW) and Selenite cystain Broth (SCB) for enrichment and incubated at 37°C up to 6 hours. Afterward, 0.1 ml of each sample from the broth was introduced on selective media such as Salmonella, Shigella agar and the Thiosulfate Citrate Bile Salt Sucrose agar media respectively and plates were incubated at 37°C for 24 hours. Confirmatory Biochemical tests. For the final identification of all isolates, several biochemical tests were performed including the triple sugar iron test, motility indole urease test, methyl red test, Voges Proskauer test, indole utilization test and the oxidase test.
Determination of antimicrobial susceptibility of the isolates. The pathogenic isolates were examined for antibiotic susceptibility traits (either drug resistant or sensitive) by disc diffusion assay on Mueller-Hinton agar (Difco, Detroit, MI) against commonly used antibiotics following the standard protocol. Lawns of bacterial suspensions including Escherichia coli, Pseudomonas spp., Vibrio spp., Staphylococcus spp.and Salmonella spp. (turbidity compared with the McFarland standard OD600-0.5) were prepared and introduced on to Muller Hinton agar. Antibiotics used in the study included polymixin B (300 unit), Kanamycine (30 µg), methicillin (30 µg), streptomycin (10 µg), vancomycine (30 µg), gentamycine (10 µg), nalidixic acid (30 µg), azythromycine (15 µg), penicillin G (10 µg), erythromycin (15 µg), amoxicillin (30 µg), ceftriaxon (30 µg), ciprofloxacin (5 µg), ampicillin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg) and cefixime (5 µg). All plates were incubated at 37°C for 12-18 hours and examined for formation of the zone of inhibitions (mm).