Isolation of chemically resistant bacterial strains from industrially polluted water body

Isolation of chemically resistant bacterial strains from industrially polluted water body Mahmud MS Abstract Bacground: Untreated disposal of chemical waste poses serious environmental hazard. An attempt was made towards presumptive identification of the major genera of microbial contaminants found in a natural pond that receives waste from a pharmaceutical industry of Gonoshasthaya Antibiotic Ltd. during the period of 2008 to 2009. Methodology: Water sample was collected and physical parameters were determined. Results: A total of 38 bacterial isolates were found from surface water, deep layer water and sediment soil but only five isolates were found to resist up to 0.1 mg/ml of phenol. Microscopic and biochemical test of five isolates presumptively identified them Staphylococcus sp, Sporosarcina sp, Bacillus sp and members of family Enterobacteriaceae. All five isolates were resistant to Amoxycillin (30?g), Erythromycin (15?g), and Penicillin-G (10units). Conclusion: Resistance against common therapeutic antibiotics indicates possible epidemiological risk.


Introduction
About 80% people in Bangladesh lack clean, safe water because most household and industrial wastes are dumped directly in to the natural water bodies.According to Bangladesh Bureau of Statistics about 37.5% factories lack waste management and industrial waste is dumped into the water bodies without any treatment 1 .The oxygen demanding wastes are decomposed by aerobic (oxygen requiring) bacteria.The quantity of oxygen demanding wastes in water can be determined by measuring the biological oxygen demand (BOD), the amount of dissolved Oxygen needed by aerobic decomposer to break down the organic materials in a certain volume of water over a 5days incubation period at 20 0 C (68 0 F) Effect of pollutant on micro flora of an aquatic environment draws special interest of research to find out possible ways of bioremediation.

Collection of sample
A natural pond receiving waste from antibiotic industry (GAL) was chosen and 100 ml water was collected from surface layer, 50 cm deep layer and layer near sediment in sterile Duran Bottles.

Determination of Physical and Chemical
Parameters Chemical Oxygen demand (COD) of the polluted water was determined by titrating the water against 0.1 M sodium thiosulfate using 0.25 N K2Cr2O7 as the indicator 5, 6 .
Dissolved Oxygen (DO) and Biochemical Oxygen Demand (BOD) was determined by the standard 5 day BOD test (BOD5) 6, 7, 8 .Alkalinity of the waste water was titrated against 0.1 N HCl using methyl orange as the indicator 7 .The temperature and pH were determined respectively by thermometer and Mettler pH meter.

Isolation of organisms
Each specimen was cultured on nutrient agar plate and incubated at 37 0 C over night.Next day, the types of colonies were observed and recorded.Mac Conkey agar, Eosin Methylene Blue (EMB) Agar, Blood agar, K. F. Streptococcus agar (KFSA) and Mannitol Salt Agar (MSA) were used for cultivation of the suspected organisms in the sample water 9 .

Screening for phenol tolerant organism:
Two sets of test tubes were prepared with phenol containing media.One set had T1N1 (Tryptone NaCl) media with 0.1 mg/ml phenol.Another set had 0.85% NaCl solution with 0.1 mg/ml phenol.Both sets were inoculated with the isolates and incubated at 37 0 C for 3 days, the tubes were observed for growth.

Biochemical Test for Presumptive Identification
The phenol-tolerant isolates were taken for Gram

RESULT
DO, COD, Alkalinity, P H and temperature of the water was determined .The result is recorded in Table I.
The water samples were cultured on nutrient agar media and a total of 38 different isolates were found.Among them 11 isolates showed growth on T1N1 supplemented with 1% phenol and 5 isolates could grow in 0.85% NaCl solution.These 5 isolates are chosen for further experiments.The results of microscopic observation are presented in Table II.
The growth of the isolates on different cultural media is described in Table III.
Further test of hydrolysis was done for the Gram positive isolates, the result of which is presented in Table V.
ical tests, the isolates were identified with reference to Bergey's Manual of Systemic Bacteriology 12 and Bergey's Manual of Determinative Bacteriology 13 .The identity are presented in Table VI.

Discussion
The aim of this study was to assess the microbial flora of industrially polluted water and to find out the tolerance or resistance of the organisms against chemicals.A natural pond with effluent from an antibiotic industry was chosen because this effluent contains antibiotics along with other industrial chemicals 13 .Phenol was chosen because it is a common bacrtericidal agent and there is a correlation between rise of phenol resistance and antibiotic resistance 14 .The bacteria that can survive overnight in 0.1 mg/ml phenol are considered resistant to it.The five phenol-resistant isolates identified on biochemical test were common organisms of tropical water (Table IV).The member of The result of Biochemical profiling is presented in Table IV.
Depending upon the microscopic and the biochem.The resistance of these isolates against antibiotics is listed in Table VII.
Family Enterobacteriaceae (Table III Table VII).This isolate shows the highest degree of drug resistance.Such a pattern of resistance from a natural strain is uncommon since most reported strains are sensitive to Vancomycin and Erythromycin 18 .Probability of adaptation to the antibiotics present in the industrial effluent could be a reason.Detailed study of virulence and molecular characterization of antibiotic resistance factors was not within the scope of this study, but extensive research of pathogenic potential of the Bacillus sp. that are antibiotic resistant might generate new information about pollution-induced community tolerance and spread of antibiotic resistance 19, 20 .