The soil samples were collected from agricultural fields with 5-10 years history of insecticidal spray from different parts of Punjab, Pakistan. Fifty nine bacterial strains resistant to organochlorine insecticides, i.e. endosulfan and heptachlor (cyclodiene group) were isolated. They were also capable of utilizing organophosphates (Anthio, Ethion, Quinalphos), pyrethroids (Bifenthrin, Cypermethrin) and a carbamate (Carbosulfan) as a carbon source in an inorganic M-9 agar medium. Thirty five of these bacterial strains were isolated from Lahore region and twenty four from Multan region. The minimum inhibitory concentration (MIC) of endosulfan and heptachlor ranged from 2 to 10 mg/ml, while that of organophosphates (OPâ€™s) from 1 to 6 mg/ml, of pyrethroids from 2 to 12 mg/ml and of carbosulfan from 1 to 4 mg/ml. On the basis of high MICs twenty four bacterial strains were finally selected for further analysis.
The optimum pH and temperature of the selected bacterial isolates was 6.5-7.5 and 30-42Â°C, respectively. The growth patterns of the isolates were studied in different media viz., LB, M9+glucose (50 mg/100 ml), M9+endosulfan (50 mg/100 ml) and M9+heptachlor (50 mg/100ml). In LB medium typical growth patterns were observed as compared with those in M9, in which prolonged lag or stationary phases were observed. Eight isolates, based upon the various biochemical tests, were identified as members of the genus Bacillus, two each of the genus Kurthia and Caryophanon and one each of Sporolactobacillus, Derxia, Streptococcus, Aureobacterium, Alcaligenes, Terrabacter, Rizhobacter, Azomonas and Pimelobacter.
They were also tested for sensitivity to eight heavy metals. The MICs of Cd2+ varied from 300-500 Î¼g/ml, of CO2+ 1 00-200 Î¼g/ml, of Cr6+ 300-500 Î¼g/ml, of Cu2+ 350-450 Î¼g/ml, of Hg2+ 1 00-200 Î¼g/ml,. of Ni2+ 300-550 Î¼g/ml, of Pb2+ 1500-2500 Î¼g/ml and of Zn2+ 300-500 Î¼g/ml.. The antibiotic sensitivity of isolates was also checked against nine antibiotics. The isolates were resistant to ampicillin, amoxycillin, cefixime and cefotoxim but were sensitive to minocyclin. Most of the bacteria were sensitive to cefazoline except for CMBLI 62, 75, 76 and 87, to chloramphenicol except for CMBLI 43, 44, 46, 53, 55 and 65 and tetracycline except for CMBLI 38, 53 and 55.
All isolates were found to harbour plasmids of 23 Kb except for CMBLI 62, which had five plasmids of 23, 13, 6.5, 4.2 and 2.9 Kb size. All the isolates could be cured of their plasmids, which made them sensitive to insecticides mixed in the growth media. The competent cells of E. coli C600 were successfully transformed with the plasmids of isolates. The transformed E. coli could grow on M9 selective medium containing 50 mg/100 ml of endosulfan/heptachlor. The 23 Kb plasmid was later successfully recovered from the transformants. It was concluded that the gene for insecticide degradation was located on the plasmids.
The total proteins of some selected isolates grown in LB, M9+glucose, M9+endosulfan and M9+heptachlor (50 mg/100 ml) were extracted in the sample buffer and were analyzed by 12% 8DS-PAGE. More protein bands were seen in isolates grown in LB media as compared with those in M9 media. Despite the suppression of certain protein bands in isolates grown in insecticide-containing media, protein bands of 64, 62, 59,50 and 37 Kd size were present in M9 as well as in control media. The presence of protein bands in insecticide stress media implicate their role in detoxification of insecticides. The isolated bacterial strains can be employed in the microbe based bioremediation of insecticide-contaminated soil and waste water