Study area, sampling and sample processing. Four different types of tannery waste samples (tannery wastes after lather treatment, CaCO3 containing wastes, liquid tannery wastes and solid tannery wastes) and four different environmental samples (surface water from lake, dry soil sample from waste dumping area, solid wastes from dumping area and wet soil sample from waste dumping area) were randomly collected from a tannery industry located at Hazaribagh, Dhaka, Bangladesh and its surrounding environment from November 2015 to January 2016 following standard protocol. For the identification and enumeration of pathogenic bacteria and fungi, 10g or ml of each sample was blended with 90 ml of buffer peptone water (pH 7.2 ± 0.2) and diluted up to 10-6 for all the samples tested according to the standard guideline.
Isolation and identification of bacteria Estimation of total viable bacteria, Escherichia coli, Klebsiella spp., Staphylococcus spp. Bacillus spp. and Pseudomonas spp. For each of the cases, 0.1 ml of samples from the dilution (10-4 and 10-6 ) for the solid and (10-2 and 10-4 ) for the liquid was introduced on to the nutrient agar and sabouraud dextrose agar for the isolation of total viable bacteria and fungi, respectively. Likewise, each sample was introduced onto MacConkey agar, mannitol salt agar (MSA), starch agar and Pseudomonas agar for the isolation of coliforms (Escherichia coli and Klebsiella spp.), Staphylococcus spp., Bacillus spp. and Pseudomonas spp., consecutively. All the plates were then incubated at 37oC for 24 hours.
Isolation of Salmonella spp., Shigella spp. and Vibrio spp. By considering the possible occurrence of viable but non-culturable (VBNC) cells (24-28), 10 ml of sample was transferred into 90 ml of selenite cysteine broth (SCB) and alkaline peptone water (APW) for the enrichment of Salmonella spp., Shigella spp., and Vibrio spp., respectively and incubated at 37oC for 6 hours. After incubation, the samples were diluted up to 10-4 and then 0.1 ml of samples from each of the 10-2 and 10-4 dilutions were spread onto Salmonella-Shigella (SS) agar and thiosulfate citrate bile salt sucrose (TCBS) agar for the isolation of Salmonella spp. & Shigella spp., and Vibrio spp., consecutively. Plates were incubated at 37oC for 48 hours for the detection of typical colonies.
Antibiotic susceptibility test of the isolates. The standard agar-disc-diffusion method (Kirby Bauer technique) was used to examine the antibiotic a susceptibility of the isolates (either sensitive or resistance) on Mueller-Hinton agar (Difco, Detroit, MI) (24, 29-31). The antibiotic discs used in this experiment were Ampicillin (AMP, 10 μg), Imipenem (IPM, 10 μg), Azithromycin (AZI, 15μg), Penicillin (PEN, 10μg), Gentamycin (GEN, 10 μg), Streptomycin (STM, 10μg), Erythromycin (ERY, 15μg), Ciprofloxacin (CIP, 5μg), Cefixime (CFX, 5μg) and Chloramphenicol (CHL, 10 μg). The plates were then inverted and incubated at 37°C for 24 hours. After incubation, the plates were examined and the zone of inhibition was measured in mm.
Antimicrobial assay. For the determination of antimicrobial activity, modified agar well diffusion method was followed using Mueller-Hinton agar plate. Suspensions of different bacteria such as E. coli, Pseudomonas spp., Micrococcus spp., Vibrio spp., Klebsiella spp., Staphylococcus aureus, and Salmonella spp. was introduced on to the MHA agar were prepared using normal saline, consisting of 106 cfu/ml with a turbidity equivalent to that of the 0.5 mL McFarland standard, and each suspension was then subject to lawn on the MullerHinton agar (MHA) (Oxoid Ltd., Basingstoke, Hampshire, England). The wells were dug (8 mm3) on the inoculated Muller Hinton agar medium and 100μl or 11mg/ml of each sample were introduced. Normal saline was used as negative controls whereas antibiotic disk of Gentamycin (GEN, 10 μg) was used as positive control. The plates were incubated at 37oC overnight and examined for the zone of inhibition. The diameter of the inhibition zone was measured in mm using slide calipers.