Industrialization and human activities have totally turned our environment to dumping sites for wast

Industrialization and human activities have totally turned our environment to dumping sites for waste materials

1. Introduction Industrialization and human activities have totally turned our environment to dumping sites for waste materials. As a result, many water resources have been rendered unwholesome and hazardous to man and other living systems (Bakare et al. 2003). The toxic substances discharged into water bodies are not only accumulated through the food chain (Odiete 1999), but may also either limit the number of species or produce dense populations of microorganisms (Okafor 1985). Pollution of the surface and underground water wastes is widespread, thereby rendering them unsuitable for man’s use (Ajayi & Osibanjo 1981; Kakulu & Osibanjo 1992; Odiete 1999; Bakare et al. 2000; Bakare & Oyedeji 2001; Bakare et al. 2003). In addition, since many industries lack effluent treatment plants, the untreated wastes are either deposited on the ground or discharged into nearby natural water bodies (Chukwura & Okpokwasili 1997; Odiete 1999).

Industrial effluents are wastes generated by the industry during the process of drugs manufacturing. Here we identify and isolate microorganisms from effluent of the industry and also measure the quality of effluent treatment plant of ACME Laboratory.

ACME Laboratories produces almost 400 products and The ACME Agrovet & Beverage Ltd, savar, manufacture fruit juice, mineral water and many more. Discharges waste material in reservoirs situated within the area of the industry.

ACME Laboratory an industry producing (amoxicillin, ampicillin, cephalexin, cloxacillin, flucloxacillin). The waste disposal of this industry contains of a range of chemical substances including methylene chloride, isopropyl alcohol, pivalic acid, triethylamine, small amount of amoxicillin and its degradation products, ethanol acetone Cephalexin and its degradation products, ethyl acetate, methyl ketone and small amount of cloxacillin its degradation products. (Susan et al 1989, Golam Mahammed, 2005).

In this study, we undertake the microbiological assessment of the effluent of a pharmaceutical company name ACME Laboratory whose effluent is discharged through an effluent treatment plant. After treatment the treated water is reserved into a man-made lake. When it rains, the water is washed down the terrain into a stream, where as in the dry season a substantial portion of the water sinks into the ground.

Microorganisms of polluted environment bear resistant property to that pollutant. Like other normal microbes they are identified based on morphology, nutritional requirement and growth characteristic. Comparing the viability of microorganisms of polluted environment to that of natural habitat,. A chemical industry without waste treatment facility always discharges materials in natural habitat harmful to living world comprising human, animal, plants, and the important microbial community.

We assess the effect of polluting waste water and treated water from an effluent treatment plant (ETP) on microbes from a reputed industry namely ACME Laboratories Ltd. 1.1 General information: Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. Bacterial cell components have their own defense mechanism against their lethal components. .

Fig: Bacterial components of cell responsible for resistance against antibiotics ( natural ) Antibiotic resistance evolves via natural selection acting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by conjugation, transduction, or transformation. Many antibiotic resistance genes

reside on plasmids, facilitating their transfer. If a bacterium carries several resistance genes, it is called multi resistant or, informally, a superbug.

Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place.

The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.

Most of the microorganisms get mutated to antibiotic resistance under untreated condition due to their long time presence in that condition to survive

Antibiotic resistance of organisms are now creating worse situation day by day. The rate of resistance is increasing regularly. Therefore it is now creating a very critical condition to check. The increasing antibiotic resistance is a matter of concern not only in treating diseases but also in treating environment that is the waste management. There are a number of factors

that are responsible for getting antibiotic resistance among the microorganisms . some are as follows: •

The wide spread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria.

•

They are often used in animals but also in other industries which at least in the case of agricultural use lead to the spread of resistant strains to human populations.

•

In some countries antibiotics are sold over the counter without a prescription which compounds the problem. In human medicine the major problem of the emergence of resistant bacteria is due to misuse and overuse of antibiotics by doctors as well as patients.

•

Other practices contributing towards resistance include the addition of antibiotics to the feed of livestock.

•

Household use of antibacterial in soaps and other products, although not clearly contributing to resistance, is also discouraged (as not being effective at infection control).

•

Unsound practices in the pharmaceutical manufacturing industry can contribute towards the likelihood of creating antibiotic resistant strains.

•

Certain antibiotic classes are highly associated with colonization with superbugs compared to other antibiotic classes. The risk for colonization increases if there is a lack of sensitivity (resistance) of the superbugs to the antibiotic used and high tissue penetration as well as broad spectrum activity against "good bacteria".

1.2 Mechanisms Bacteria use different mechanisms to protect themselves from natural, synthetic and semi synthetic compounds that are The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are: 1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.

2. Alteration of target site: e.g. alteration of PBB—the binding target site of penicillin’s —in MRSA and other penicillin-resistant bacteria.

3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.

4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface. Some bacterial spp use it.

But it is now getting attention. They posses efflux transporters of very broad substrate specificity. Some bacteria are able to withstand synthetic and semi synthetic agents by pumping out.

2. Objective: (a) To isolate microorganisms from chemically polluted water source. (b) To isolate microorganisms from ETP to check its efficiency. (c) To characterize the isolates microbes with resistance pattern to antimicrobial drugs. (d) To indicate the spreading of drug resistant microbial strains to community endangering people’s health. (e)To focus on efficiency level of ETP of ACME Laboratories.

2.1 Outline of the study 1. Objective Selection 2. Sample collection 3.simple analysis like alkality, ph, temperature, etc 4.BOD 5.COD 6. Microbiological analysis of sample a. Microscopic examination

b. Isolation of microbes by culturing the sample c. Cultural characterization of the isolates d. Colony characters on solid media e. Morphological and staining properties f. Biochemical properties g. Antibiotic sensitivity testing of the isolates h. Survival study of the isolates with polluted water 3. Materials and Methods The duration of the study was from May 2010 to October 2010 at Gonoshasthaya Vaccine Research and Diagnostic Laboratory (GVRL), Department of Microbiology, Gono Bishwabidyalay. MacConkey agar was purchased from Oxoid Ltd., England and Tropic soy agar for blood agar plates was from Becton Dickinson and Company, USA. Mueller Hinton agar was purchased from Sanofi Diagnostics, Pasteur, France. Antibiotic discs amoxicillin (30 μg), were purchased from Becton Dickinson and Company, USA. Ciprofloxacin (5 μg), cotrimoxazole (25 μg), tetracycline (30 μg) and gentamicin (120 μg), nitrofurantoin (30μg) were from Oxoid Ltd. UK. Nalidixic acid (30 μg). Erythromycin Diagnostics, UK.

3.1 Materials Table-I Antibiotic discs used in the study:

were from Mast

Sheep blood was collected from animal house of GVRL. Other ingredients were of appropriate analytical and commercial standards.

Sl. No. 1 2 3 4 5 6 7 8 9 10 13

Antibiotic Amoxicillin Ciprofloxacillin Cotrimoxazole Gentamicin Imipenem Nalidixic acid Nitrofurantoin Tetracycline Cephalexin Penicillin G Erythromycin

Letter Code A CIP SXT CN IMI NA NI TE CFX PG E

Quantity 30 μg 5 μg 25 μg 120 μg 10 μg 30 μg 300 μg 30 μg 30 μg 10 unit 15

Source Becton Dickinson, USA Oxoid Ltd., UK Oxoid Ltd., UK Oxoid Ltd., UK Mast Diagnostics, UK Mast Diagnostics, UK Oxoid Ltd., UK Oxoid Ltd., UK Oxoid Ltd., UK Mast Diagnostics, UK Mast Diagnostics, UK

Both qualitative and quantitative microbiological analyses were performed. For quantitative routine examination, the samples were mixed thoroughly before plating. Inoculation on media was done with pre-calibrated platinum loop to deliver a measured quantity (1.0 μl) of sample.

For culturing MacConkey-agar and 5% sheep blood-agar plates were used that can support the growth of most Gram-negative bacilli and staphylococci (Baron and Finegold, 1990). The inoculated plates were incubated at 37ºC overnight. Plates with tiny colonies or no growth were extended to further incubation up to 48 hour.

For qualitative analysis, identification of organisms was done by conventional methods through culturing of samples followed by biochemical tests (Monica, 1984). Antimicrobial sensitivity was performed by disc diffusion method on Mueller-Hinton agar plates for organisms other than streptococci which needed blood agar plates.

Glass wares and other materials: The different types of sterilized glass ware and materials are used.

1.

Experimental test tube.

2.

Durham` s fermentation tube.

3.

Stopper of test tube.

4.

Petri dish.

5.

Conical flask.

6.

Pipette.

7.

Slide.

8.

Microscope

9.

Immersion oil.

10.

Cotton swab

11.

Thermometer.

12.

Beaker

13.

Jar, Cylinder.

14.

Electric Balance.

15.

Toothpick

16.

Spirit lamp and

17.

Bacteriological loop

18.

Bacteriological straight wire etc.

3.2 Methods 3.2.1 Sample collection: Sample was collected from the untreated water reservoir of ACME Laboratories and from the pond where treated water was discharged. Waste water samples were collected from surface, deep layer and sediment of the reservoir and tasted for the presence of microorganisms.

3.2.2 Sample collation site Samples were collected from ACME Laboratories.

Production plant 1)

waste water reservoir (sampling point-

ETP (sampling point-2)

Treated water reservoir Fig: overall process of ACME Laboratories 3.2.3 study site This study was carried out in the Department of Microbiology, Gono Bishwabidyalay (University ).

3.3 Chemical parameters of water 3.3.1 COD of water (a) (b) (c) (d) (e) (f) (g)

Poured 50 ml of water sample in a conical flask (100 ml capacity).

Similarly, took 50 ml distilled water in a flask.

Poured 5 ml K2Cr2O7 solution separately in both flasks.

Incubated the flasks at 1000 C for one hour keeping in a water bath.

Thereafter, removed the flasks to cool for 10 minutes.

Mixed 5 ml KI solution, and 10 ml of H2SO4 solution in each flask.

Transferred 0.1 M sodium thiosulfate solution in burette fitted in titration assembly, and titrated with both the samples in flasks till pale yellow color disappears. In each case noted the amount of sodium thiosulfate solution used. (h) Added 1 ml of starch solution to both the flasks. Color turned blue. (i) Again titrated with sodium thiosulfate as above till complete disappearance of blue color.

3.3.2 DO of water

(a) Collected water sample in a glass bottle (250 ml) in such a way that water bubble should not come out. (b)Pipette separately 2 ml of manganese sulfate and 2 ml of alkaline iodine-azide solutions. (c)Added these solution in succession at the bottom of bottle and placed the stopper of bottle (d)Shook the bottle upside down for about 6-8 times. There developed brown precipitate. (e)Left the bottle for a few minutes, the precipitate settled down. (f)Added 2 ml of concentrated H2SO4 in the bottle. Shook properly so that brown precipitate might dissolve. (g)Took a clean flask and pour 50 ml of this water sample. Titrated it against 0.025 N sodium thiosulfate solution taking in a burette until pale straw color develops. (h)Add 2 drops of starch solution on the flask. Color of contents changed from pale to blue. (i)Again titrated against thiosulfate solution until the blue color disappeared.

3.4.1 Alkalinity of water (a) Took 50 ml of water sampler in a conical flask (100 ml capacity). (b) Added a few drops of phenolphthalein indicator.( If color of the water does not change, it means that phenolphthalein alkalinity is nil due to absence of carbonates in the water sample. Moreover, if there develops pink color, determine phenolphthalein Alkalinity). (c) Poured 0.1N Hcl solution in burette and titrated with water sample. Noted the end point of when pink color become colorless. (d) Took another 50 ml of water in flask and added 2-3 drops of methyl orange in to it. Color turned to orange. (e) Transferred 0.1N Hcl solution in to burette in titration assembly and titrated with the water sample. Methyl orange added until yellow color change to pink. Noted the end point.

3.4.2 PH of Water The PH of water sample was determined correctly by PH meter.

3.4.3 Temperature of water The temperature of water sample was determined correctly by thermo- meter.

3.5 Isolation of viable organisms.

Each specimen was streaked on nutrient agar plate and incubated over night. Next day, the types of colonies were observed and recorded.

3.6 Screening for phenol tolerant organism.

Two sets of test tubes were prepared with phenol containing media. One set had T 1N1 media with 1% phenol. Another set had distilled water with 1% phenol. Both sets

were inoculated with the isolates and incubated at 37 0 C for 3 days, the tubs were observed for growth.

3.7 Gram Staining. (a) Obtained clean glass slides (b) Using sterile technique, prepared a smear of each of the organisms. Did this by placing a drop of water on the slide, and then transferring each organism separately to the drop water with a sterile, cooled loop. Mixed and speeded organism by means of a circular motion of the inoculating loop. (c) Allowed smears to air-dry and then heat fixed in the usual manner. (d) Gently flooded smears with crystal violet and let stood for 1 minute. Gently washed with tap water. (e) Gently flooded smears with Gram’s iodine mordant and let stood for 1 minute.

Gently washed with tap water. (f) Decolorized with 95% ethyl alcohol. Gently washed with tap water. (g) Counterstain with safranin for 45 sec. (h) Gently washed with tap water. (i) Examined under oil immersion.

3.8 Spore Staining. (Schaeffer-Fulton Method ) (a) (b) (c) (d) (e) (f) (g) (h)

Obtained one cleaned glass slide.

Made smears in the usual manner using sterile technique.

Allowed smear to air dry, and heat fixed in the usual manner.

Flooded smear with malachite green and placed on top of a beaker of water sitting on a warm hot plate. Allowing the preparation to steam for 2 to 3 minutes. Prevent the stain from boiling by adjusting the hit plate temperature.

Removed slide from hot plate, cool, and wash under running tap water.

Counterstain with safranin for 30 sec.

Washed with tap water.

Examined under oil immersion.

3.9 Biochemical test 3.9.1 .Catalase Test a) A colony of the bacteria from a plate was picked up and transferred on a glass slide in a drop of water. b) A few drops of 3% H 202 was placed (dilute 30% commercial solution 1: 10) over the slide. c) Production of gas bubbles (released oxygen) indicated appositive reaction.

3.9.2 Oxidase Test

a) A portion of the test organism was picked up from agar plate by means of a sterile wooden-pick. b) Streaking on to the filter paper soaked with the oxidase reagent. c) Formation of a dark purple color developed within 5-10 seconds indicated positive for oxidase 3.9. 3. Triple Sugar Iron (TSI) agar Test a) A loop of bacteria was spread across the surface of the agar. b) A needle of bacteria was inserted (stabbed) into the bottom (butt) of the tube. c) The tubes were kept at 37° C for 24 hours for incubation. d) The tubes ere examined.

3.9.4 Motility Iodole and Ornithine decarboxylate ( MIO) Test a) Inoculated with 2-3 similar colonies from an over night growth, by stabbing with a straight needle to over half the depth. b) The tube was inoculated at 37° C for 24 hours with loosen caps. c) Examined by naked eyes for motility and ornithine decarboxylate.

3.9.5 Methyl red (MR) Test (a) Sterile MR-VP broth was inoculated with the test organism and following incubation at 37° C for 24 hours. b) Few drops of methyl red solution were added. c) A distinct red color indicated MR positive test while yellow or orange color indicated a negative result.

3.9.6 Voges proskaur (VP) Test a) Sterile MR-VP broth was inoculated with the test organism and following incubation at 37° C for 24 hours. b) After incubation, 5 rops of naphtha solution and 5 drops of KHO solution were added. c) The development of a bright red or pink-red color was recorded as a positive result.

3. 9.7 Indole Test. a) Tryptophan containing broths were inoculated with bacteria. b) The tubes were incubated at 37° C for 24 hours. c) Added 0.5 ml of the Kovac's reagent after the bacterial growth. If indole positive, after 2 minutes a red color ring appeared at the junction of medium and reagent in the tube 3.9.8 Citrate Utilization Test. a) A loop of bacteria was spread across the surface of the agar.

b) A needle of bacteria was inserted (stabbed) into the bottom (butt) of the tube. c) Then the tubes were kept at 37° C for 24 hours for incubation. d) Finally the tubes were examined.

3 .9 .9 UreaTest. a) The organisms were inoculated in tubes containing urea slant. b) The tubes were incubated at 37° C for 24 hours. c) Bright red color indicated positive result.

3.9.10 Carbohydrate Fermentation.(sucrose, lactose, dextrose) (a) Phenol red broth medium containing inverted Durham’s tube was inoculated with the test organism. (b) Incubated at 370 C for 24 hours. (c) Chang in color indicated acid production while formation of bubble in the Durham’s tube indicated gas production.

3.9.11 Nitrate Reduction Test a) Sterile nitrate broth was inoculated with the test organism. b) Incubated for 24 hours at 37° C. c) Following incubation one drop of sulfanilic acid and one drop of napthalamine were added. d) Formation of a red color indicated reduction of nitrate. If no color developed, zinc dust was added and absence of any color again indicated a positive result.

3.9.12 Mannitol Fermentation Test. a) one loop of test organism was inoculated in the mannitol broth. b) Incubated the tube at 37° C for 24 hours. c) The tubes were examined, in case of producing acid, the color of the medium would be changed from purple to grayish yellow. The medium remaining purple color indicated that no acid was produced.

3.9.13 6.5% Nacl test a) The organisms were in inoculated in 6.5 % Nacl broth. b) The tubes were incubated at 37° C for 24 hours. c) The results were observed.

3.10 Antibiotic Sensitivity Assay Of Bacterial Isolates.

Bacterial susceptibility to anti microbial agent was determined in vitro by using the standardized agar disc-diffusion method known as the Kirby Bauer ( Barry and Thom berry, 1985 ). Antibiotic and the disc potencies used were following

Imipenem (10 µg), Azithromycin (15µg), Amoxicilin (30 µg), Tetracyclin (30 µg), Nalidixic Acid (30 µg), Erythromycin (15 µg), Vancomycin (30 µg), Cephalexin (30 µg), Amikacin (30 µg), Penicillin-G (10 units). (a) Labeled the covers of each of the agar plates with name of the test organisms was inoculated. (b) Using sterile technique, inoculated all agar plates with their respective test organisms as follow: 1) Dipped a sterile cotton swab into a well mixed saline test culture and removed excess inoculums by pressing the saturated swab against the inner wall of the culture tube.

2) Using the swab, streaked the entire agar surface horizontally, vertically, and around the outer edge of the plate to ensure a heavy growth over the entire surface. (c) Allowed all culture plates to dry for about 5 minutes. (d) Distributed the individual antibiotic discs at equal distance with forceps dipped in alcohol and flamed. (e) Gently pressed each disc down with the wooden end of the cotton swab or sterile forceps to ensure that the discs adhered to the surface of the agar. (f) The plates were then inverted and incubated at 370 C for 24 hours. (g) After incubation, the plates were examined and the diameter of the zones of complete inhibition were measured in mm. (h) The zone diameter for individual anti-microbial agents were used to determine susceptible, intermediate, and resistant categories by referring to an interpreting table ( Barry and Thom berry, 1985 ).

3.11 Observation of cultural characteristic.

3.11.1 Blood Agar.

Each isolate was streaked on blood agar and incubated at 37 0C for over night. Next day demonstrated the patterns of haemolysis produced by organisms.

3.11.2 Eosin Methylene Blue (EMB) Agar.

Each isolate was streaked on EMB agar plate and incubated at 37 0C for over night. Next day demonstrated morphological characteristics of the bacterial colonies.

3.11.3 MSA Each isolate was streaked on MSA plate and incubated at 37 0C for over night. Next day demonstrated morphological characteristics of the bacterial colonies.

3.12 Oxygen Utilization Test.

(a) Using sterile technique, inoculated each experimental organism by introducing two drops of the culture from a sterile pipette into the appropriately labeled tubes of molten agar. (b) Vigorously rotated the freshly inoculated molten infusion agar between the palms of the hands to distribute the organisms. (c) Placed inoculated test tubes in an upright position in the ice water bath to solidify the medium rapidly. (d) Incubated the tubes at 370C for 24 hours.

4.1.1 Result of chemical parameter of waste water and treated water.

DO, COD, Alkalinity, PH, and temperature of the water was determined .The result is recorded in below table.

Table No-1 Source DO BOD COD PH Temperature Waste 2.5mg/L 750mg/L 1190.0mg/L 6.5 35.00 C water Treated 6.61mg/L 23.85 19.22 mg/L 7.3 24.80 C water 4.1.2 Result of isolation of viable organisms.

Each water sample was streaked on NA plate and types of colonies were observed and record.

The result is recorded in below table.

Table No-2 Specimen Nutrient Agar Plate Surface water Different colonies of 17 morphological types were found code numbered A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11,A12,A13,A14,A15,A16,A17.

Deep water Different colonies of 12 morphological types were found code numbered E1,E2,E3,E4,E5,E6,E7,E8,E9,E10,E11,E12.

Sediment water Different colonies of 10 morphological types were found code numbered H1,H2,H3,H4,H5,H6,H7,H8,H9,H10.

[All colony types were repeatedly sub cultured in nutrient agar to get isolated pure culture of each type of colony ] .

4.1.3 Screening.

All colony types were inoculated in T 1N1 broth, T1N1 supplemented with phenol and diluted phenol solution and there growth was observed for 3 days. The result is recorded in below table.

Table No-3.

Media

composition

Isolates Code

Growth after 3 days.

T1N1 Supplement ed phenol

Nacl-10.gm Trepton10.gm Phenol-10gm Distilled water-1000ml

A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11,A12,A13,A 14,A15,A16,A17.

A1, A3, A7

E1,E2,E3,E4,E5,E6,E7,E8,E9,E10,E11,E12.

E2, E5, E6, E9 H3, H4, H7

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10.

Table No-4.

Media Compositio Isolates Code n Dilute 0.5gm A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11, phenol phenol A12,A13,A14,A15,A16,A17. solution crystal in 5ml water. E1,E2,E3,E4,E5,E6,E7,E8,E9,E10,E11,E12.

(100Âľg/ml) H1, H2, H3, H4, H5, H6, H7, H8, H9, H10.

Growth after 3 days.

A1, A3, A7 E2, E5, E6, E9 H3, H4, H7

4.1.4 Result of Gram staining.

Physical, Staining, and cultural properties of the bacterial strains isolated from industrially polluted waste water are recorded in below table.

Table No-5 Isolates Code

Gram Reaction

Call

A1 A3

Gram (-) Ve Gram (+) Ve

Morphology Rod Rod

A7 E2 E5

Gram (-) Ve Gram (-) Ve Gram (-) Ve

Rod Rod Rod

E6 Gram (+) ve E9 Gram (-) ve H3 Gram (+) ve H4 Gram (-) Ve H7 Gram (-) Ve 4.1.5 Result of Spore staining.

Table No-6 Isolates A1 A3 A7 E2 E5

Rod Rod Cocci Rod Rod

Presence of spore Non spore Non spore Non spore Non spore Non spore

E6 E9 H3 H4 Isolate no.

H7 A1, H4 A3, E6 A7 E2 E9 E5, H7

H3

Nutrient agar

Blood agar

Thick, white, glestining growth Slightly elevated, Blue grey spreading colonies White, moist, glistening growth White, translucent, raised growth Moist, greenish, spreaded colonies Abundant, opaque golden growth

Alpha haemolytic Beta -haemolytic Beta haemolytic colonies White, dry type colonies Alpha haemolytic colonies Beta haemolytic colonies Beta haemolytic colonies

Non spore Non spore Non spore Non spore MacConkey Non spore agar

EMB

Thick, No growth mucoid, pink colonies No growth Circular spread colonies Pink, umbunate colonies Pink, moist colonies

Reddish type No growth growth Green metallic sheen Growth found

No growth

Raised colonies

Spread growth

No growth

No growth

No growth

Yellow shiny colonies

4.1.6 Colony character of the isolated organisms in different media: Table No-7 4.1.7 Biochemical characteristics.

MSA

No growth

Isola tion No:

L a ct o s e

D e x t r o s e

Name of biochemical tests S T M M V I u S I R P n c I O d r o o l s e e

C it r a t e

U r e a s e

Nit rate Re duc tion

Ca ta la sa

Oxi da se Presumptive Organism

A1

A.S/ A A A A.B G G G H2S-

M+ O+

-

+ -

+ - +

+

-

Enterobacter spp

A3

-

A.S/ A A A.B H2S-

MO+

-

+ -

-

- +

+

-

Bacillus spp

-

A.S/ A A A.B G G H2S+

M+ O+

+

-

+ -

+ +

+

-

Proteus spp

MO+

+

-

+ -

- +

+

-

Escherichia coli

M+ O+

-

-

-

+ - +

+

-

Pseudomonas spp.

MO+

-

+ -

-

- +

+

-

Bacillus spp

MO+

-

-

+ + +

+

-

Klebsiella spp

A7

E2 E5 E6 E9

A.S/A A A A .B G G H2SNo - - - Chang e A.S/ - A A A.B H2SA.S A A A /A.B G G G H2S-

-

Samp le

Isola tion no:

H3 H4 H7

Isola te no.

L D a e ct x o t s r e o s e A A

Name of Biochemical Tests S u c r o s e

TSI

A A.S/ A.B H2SA A A No G G G Cha nge - - - No Cha nge

MIO

M V I R P n d o l e

C it r a t e

U r e a s e

Nit rat e Re du cti on

Ca ta la sa

Oxi da se

M+ O+

+

-

-

-

+

+

-

Staphylococcus aureus

M+ O+

-

+ -

+ -

+

+

-

Enterobacter spp.

M+ O+

-

-

+ -

+

+

-

Pseudomonas spp.

-

-

Presumptive Organism

4.1.8 Identification of isolated organisms studied for the isolates from the polluted waste water for identification at isolate at The staining properties, colonial morphology, and biochemical properties were genera level.

Isolate code

Presumptive organisms

A1

Enterobacter spp

A3

Bacillus spp

A7

Proteus spp

E2

Escherichia coli

E5

Pseudomonas spp.

E6

Bacillus spp

E9

Klebsiella spp

H3

Staphylococcus aureus

H4

Enterobacter spp.

H7

Pseudomonas spp.

4.1.9 Result of sensitivity test Sensitivity of the isolated bacterial strains was done on Mueller Hinton agar plate medium with the standard antibiotic discs from commercial source. Zone of inhibition was determined by referring to an interpreting table ( Barry and Thornsberry, 1985 ).

Antibiotic sensitivity test for Enterobacter spp Isolation code A1 Sensitive Intermediate Name of (S) (I) Antibiotic Zone Zone (S) size (I) size (mm) (mm) Amoxicillin Cefalexin Tetracycline I 13 Gentamycin Cotrimoxazole Ciprofloxacin Erythromycin Nalidixic acid Nitrofurantoin

Resistant (R) (R) R R R R R R R

Isolation code H4 Sensitive Intermediate (S) (I) Zone Zone (S) size (I) size (mm) (mm) S 25 S 22 S 18 S 20 S 30 S 25

Resistant (R) (R)

R I

13

Antibiotic sensitivity test for Bacillus spp Isolation code A3 Sensitive Intermediate Name of (S) (I) Antibiotic Zone Zone (S) size (I) size (mm) (mm) Amoxicillin Cefalexin Tetracycline Gentamycin Cotrimoxazole Ciprofloxacin Erythromycin Nalidixic acid Nitrofurantoin S 20

Resistant (R) (R) R R R R R R R

Isolation code E6 Sensitive Intermediate (S) (I) Zone Zone (S) size (I) size (mm) (mm)

Resistant (R) (R) R R R R R R R R R

g ta n rc e P

Sensitivity

Intermediate

Resistance

100 80 60 40 20 0

Name of Antibiotic Fig. 2: Antibiotic pattern of Bacillus

Antibiotic sensitivity test for Proteus spp

Name Antibiotic

Isolation code A7 of Sensitive (S)

Amoxicillin Cefalexin Tetracycline Gentamycin Cotrimoxazole Ciprofloxacin Erythromycin Nalidixic acid Nitrofurantoin

Intermediate (I)

(S)

Zone (mm)

S

15

size

(I)

Resistant (R) Zone size (R) (mm) R R R R R R R

S

20

Antibiotic sensitivity test for Escherichia coli

Name Antibiotic

Isolation code E2 of Sensitive (S)

Amoxicillin Cefalexin Tetracycline Gentamycin Cotrimoxazole Ciprofloxacin Erythromycin Nalidixic acid Nitrofurantoin

(S)

Zone (mm)

S

17

Antibiotic test for Klebsiella spp Name

Intermediate (I)

of Isolation code

size

(I)

Resistant (R) Zone size (R) (mm) R R R R R R R R

Antibiotic

E9 Sensitive (S) Isolation code Zone (S) size E5 (mm) Sensitive Intermediate Amoxicillin S 23 Name of (S) (I) Cefalexin S 25 Antibiotic Tetracycline S 20 Zone Zone (S) size (I)18 size Gentamycin S (mm) (mm) Cotrimoxazole Ciprofloxacin Amoxicillin S 25 Erythromycin 23 Cefalexin S 30 Nalidixic acid Tetracycline S 20 Nitrofurantoin Gentamycin S 21 Cotrimoxazole S 20 Ciprofloxacin S 27 Erythromycin S 25 Nalidixic acid Nitrofurantoin S

Antibiotic sensitivity test for Pseudomonas spp

Intermediate Resistant (I) (R) Isolation code (I) Zone size (R) H7 (mm) Resistant Sensitive Intermediate (R) (S) (I) Zone Zone (R) (S) size (I) size (mm) R (mm) R I

R

14

R R

Resistant (R) (R) R R R R R R R R R

Name Antibiotic

Isolation code H3 of Sensitive (S) (S)

Amoxicillin Cefalexin Tetracycline Gentamycin Cotrimoxazole Ciprofloxacin Erythromycin Nalidixic acid Nitrofurantoin

Intermediate (I) Zone (mm)

size

(I)

I I

Antibiotic sensitivity test for Staphylococcus aureus

Resistant (R) Zone size (R) (mm) R R R R R 12 R 14 R

Fig. 1: Growth of E. coli in EMB agar, confirmed with green metallic sheen

Fig. 2: Growth of E. coli on MacConkey Agar

Fig.3: Growth of Klebsiella on MacConkey

Fig. 4: Swarning Proteus on Blood agar

Fig. 5: Pseudomonas showing Haemolysis on Blood Agar

Fig. 6: Steaphylococcus on MSA

Fig. 7: Catalase test ( right shows positive and left is negative )

Fig. 8: Oxidase Test ( right shows positive and left is negative )

Fig.9: TSI Test ( Left shows color change of butt and showing H2S production Middle is showing acid slant/ acid butt with gas production, right is no change)

Fig. 10: MIO Test ( left is control, second tube is M+O+, third tube is M-O+ and the fourth is negative)

Fig. 11: MR- VP Test ( Left is control, Middle is MR positive And right is VP negative)

Fig. 12: Indole Test ( Left is control, middle is positive and right is negative )

Fig. 13: Citrate utilization ( left is negative and right is positive )

Fig. 14: 6.5% NaCl Test ( left is negative and right is positive )

Fig. 15: Mannitol fermentation test ( left is control, middle is negative and right is positive )

Fig. 16: Nitrate reduction test (left is control, second and third is positive and right is negative)

Fig: 17. Urea test (right is control middle is negative and left is positive )

Fig.18: multi drug resistant pattern of an isolates

Fig. 19: moderate sensitivity of an isolate

Fig. 20: gram negative bacilli

Fig. 21: gram positive cocci Discussion: Emerging antibiotics resistance of microorganisms is now a burning questions. The increasing resistance rate to various antibiotics is now creating problems.

In industrial site it is a problem to manage the waste material that is very harmful for the environment as well as for all living creatures and for our dearest world As we are the best creature of God we should try to save our environment for our existence.

For this reason waste management is very important.

It is expected from the industrial plants that they must have industrial plant(ETP). So that their treated material(waste) which is released to the environment do not cause any harm to the environment components.

In my present studies, I took sample from ACME Laboratories the sample is taken from both raw west water and ETP. A total number of 39 bacterial strains were isolated from the waste water reservoir The samples were collected from surface layer, deep layer and sediment. with cell morphology, staining, biochemical tests were performed for identification of the isolates at genus level and 10 isolates were identified.

A number of pathogenic organisms were detected from the raw waste water they are Enterobacter spp, Bacillus spp, Proteus spp, Escherichia coli, Pseudomonas spp, Klebsiella spp, Staphylococcus spp,.

When the isolated organisms were tested their reaction to certain antibiotics a tremendous picture was focused. Pseudomonas and bacillus were totally resistant to normal antibiotics.

Staphylococcus aureus were moderately resistant, Escherichia coli was getting multi drug resistance, proteus was superbug and emerging resistance was found in klebsiella.

All of the isolates are dangerous not only for human but also for other living creatures.

Proteus infections of seven days can lead for death. It is responsible for kidney failure and infection.

Staphylococcus aureus cause weaken immunity to the human body.

Escherichia coli is generally opportunistic and very severe. This infection rise when body’s defense mechanism faces many other strong infections.

Bacillus and klebsiella spp cause various types of respiratory problems not only in human beings but also in other birds and fishes.

We can easily imagine how dangerious the matter could be if they persist in our ecosystems, because if one component of ecosystem is in problem the whole system will suffer as each component is crucially dependent on the other components.

Then second sample from ETP when tested gave satisfactory result. it contain a very low level of microorganisms which is very normal. it includes a little amount of coliforms which is safe for environment. Simple analysis result was also satisfactory. the pH, temperature, turbidity, was of enough standard. The values were ph- 7.3, temperature 24.8 0 C, BOD 23.85mg/L, COD 19.22mg/L, DO 6.61mg/L. So we can say that if the treated water is released in nature it will not be harmful to environment.

This comment was done on the basis of microbiological tests of the treated water.

The same picture has been confirmed when the treated water reservoir was visited the natural scenario was excellent. There were phytoplanktons in the upper layer of the pond. The water was enough transparent and also live fishes were seen in the pond.

So it is easily understood that if the water is released to outside river or land the water will not do any harm to environment.

As ACME Laboratories is one of the most reputed ,largest and leading pharmaceuticals of Bangladesh, expectations are high to do their duties and responsibilities to the society and country.

It is a matter of great satisfaction that they are performing their social responsibilities very sincerely.

Conclusion: In this era of industrialization it is the demand of time to implement ETP in full force . it will be a matter of joke if we ignore this. But it is a matter of sorrow that most industries do not have ETP. The waste from the industries are directly thrown away to nature and the waste pollutes water source i.e. rivers, lakes, agricultural lands. The fishes are contaminated from the polluted water source. It causes various lethal disease to human .It creates an unbearable damage to the environment .This mistreatment is one of the most responsible factors of increasing global warming. This is a punishable crime. Again untreated waste cause sterility to the agriculture land slowly.

So we can say thanks to the authority of ACME Laboratories as they have taken the right decision to save the nature. Other should take this important decision within time in hand.

Only we i.e. the humans have the awareness and should take steps can save the environment from destruction.

Our government should take more necessary steps to check whether ETP is running within the industry or not and what is the efficiency level of ETPs. The above study was performed in a small scale of time with just two samples from an industry. A more and deep study is suggested with more samples from different industry.

References Atlas RM. Principles of Microbiology. Missouri, St. Louis, USA: CV Mosby Company, 491 (1995).

ASM COLLOQUIUM REPORT (1999) Antimicrobial Resistance, An Ecological Perspective. American Society for Microbiology, Washington DC, pp. 1–14.

Ahmad, S. & Yadava, J.N.S. 1979 Rapid detection of b-lactam antibiotic resistance among clinical isolates of Escherichia coli. India Veterinary Medical Journal 3, 256–259.

Ajayi, S.O. & Osibanjo, O. 1981 Pollution studies on Nigerian Rivers. II: water quality of some Nigerian rivers. Environmental Pollution. (Series B) 2, 87–95.

American Public Health Association. 1985 Standard methods for the examination of water and waste water, 16th edn. 1268 pp. Washington DC: APHA Inc. ISBN 0-87553131-8.

Bakare, A.A. & Oyedeji, S.I. 2001 Genotoxicity of leachates from a rural waste dump using two bioassays. Journal of Nigerian Society for Experimental Biology 1 11–21.

Bakare, A.A., Lateef, A., Amuda, O.S. & Afolabi, R.O. 2003 The Aquatic toxicity and characterization of chemical and microbiological constituents of water samples from Oba River, Odo-oba, Nigeria. Asian Journal of Microbiology, Biotechnology and Environmental Sciences 5, 11–17.

Bauer, A.W., Kirby, W.M.M., Sherris, J.C. & Turck, M. 1966 Antibiotic susceptibility testing by a standard single disc diffusion method. American Journal of Clinical Pathology 45, 493– 496.

Buchanan, R.M. & Gibbons, N.E. (eds). 1974 Bergey’s manual of determinative bacteriology. 8th edn. Baltimore: The Williams and Wilkins Company. ISBN 0-68301117-0.

Cappuccino, Sherman; Microbiology, A Laboratory Manual, Seventh edition Chukwura, E.I. & Okpokwasili, G.C. 1997 Impact of Brewery Wastewater on Recipient Aquatic Environments. In Biotechnology for Development in Africa: Proceedings of an International conference organized by Foundation for African Development through International Biotechnology (FADIB) held at Enugu, Nigeria, 9–13 Feb, 1997, eds. Okafor, N., Okereke, G., Miambi, E. & Odunfa, S. pp. 225–233. Enugu: Ochumba Press Ltd. ISBN 978 2791 19-9.

"Drug Resistant Infections: Riding Piggyback". The Economist. November 29, 2007. http://www.economist.com/displaystory.cfm?story_id=10205187&fsrc=RSS. E Jawetz, JL Melnick, EA Adelberg (1987) Enteric Gram negative rods in review of Medical Microbiology 17th Edition-Appleton and Lange Norwalk, Connecticut/ LosAltos,California USA.Pp 233-235.

The Journal of Antibiotics 63, 423-430 (August 2010) | doi:10.1038/ja.2010.62

Goossens H, Ferech M, Vander Stichele R, Elseviers M (2005). "Outpatient antibiotic use in Europe and association with resistance: a cross-national database study". Lancet 365 (9459): 579–87. doi:10.1016/S0140-6736(05)17907-0. PMID 15708101.

Golam Mohammed 2002, Removal of organic wastes from effluent of pharmaceuticals industries by photo chemical degradation on TiO2 and also by adsorption, filteration on hydrate ferric oxide (HFO) and manganese hydroxide coated porous and Mesoporous silicate and aluminates surface in Environmental Research Laboratory Department of chemistry Jahangirnagar University, Saver, Dhaka,Bangladesh.Pp 46, 75.

Gerad J. Tortora, Berdell R. Funke, Chemisty L. case (1998) cell wall and the Gram stain mechanism in Microbiology an introduction 6 th edition-Addision Wesly Longman, Inc.California, USA. Pp 234-235.

McARTHUR JV and TUCKFIELD RC (2000) Spatial Patterns in Antibiotic Resistance among stream bacteria: Effects of industrial pollution. Appl. Environ.Microbiol. 66 (9) 3722– 3726.

Michel J.Pelczer, Jr, E.C.S Chan and Noel R.Kriez (1998) Aquatic Microbiology, in Microbiology, 5th edition, McGraw-Hill company, Toyo, Paris, Pp 569-576.

M Alamgir Hossin, ANM Fakhruddin, Sirajul Islam Khan. BGMS. Volume 13, Number 01, March 2007. Article of the public Health at Stake by the Water pollution of peripheral river System of Dhaka. Bangladesh Journal of Medical science.Pp 44-46.

N Nahar, ANM Fakhruddin and H Rashid.Volume 21,Number 2, December 2004, Bangladesh Journal of Medical science. Aromatic Hydrocarbon-Degrading Bacteria in Industrial Chemical Water Contaminated Sites Near Dhaka City. Pp68-71.

James G.Cappuccion, Natalie Sherman (1996) MR-VP test in Microbiology A Laboratory Manual, 7th Edition, Addition-wesley, Tokyo, Japan, Pp 163-164.

R.C. Dubey, D.K. Maheshwari (2002 Practical Microbiology,1st edition.India.

Susan Sudavary, Maryadele J. O Neil, Ann Smith, Patricia E. Heckelman, An Encyclopedia of chemicals, Drugs, And Biological, in the Merck Index, 11th edition. Merck and CO, Inc. Rahway, Newjersey, USA.Pp 91, 303,820,1193, 1521, 954, 593, 11, 387, in Serial Nos of 58,610, 1971,2414,3713,3716,5095,5096,5942, 7482,9581.

WHO(January 2002). "Use of antimicrobials outside human medicine and resultant antimicrobial resistance in humans ". World Health Organization. http://www.who.int/mediacentre/factsheets/fs268/en/index.html WHO in standard operating procedure, Guidelines for Drinking water quality 2 nd edition Volume 1,2,3. World Health Organization, Geneva Pp 51-72.

Appendix-1 Composition of the media used: Nutrient Agar Peptone Bacto beef extract NaCl Agar Distilled water PH

Grams/Liter 5.0 3.0 5.0 15.0 1000 ml 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Nutrient Broth Peptone NaCl Beef Extract Yeast Extract PH Distilled water

Grams/Liter 5.0 5.0 1.5 1.5 7.4 1000 ml

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Mac Conkey agar

Grams/Liter

Bacto Peptone 17.0 Proteas Peptone 3.0 Lactose 10.0 Bile Salt 1.5 Agar 15.0 Neutral red 0.03 Crystal violet 0.001 Distilled water 1oooml PH 7.1 Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Eosine methylene blue (EMB) agar Peptone Lactose K2HpO4 Eosin Methylene blue Agar Distilled water PH

Gram/Liter 10.0 10.0 2.0 0.4 0.065 20.0 1000ml 6.8

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Blood Agar Tryptone Peptone Yeast extract NaCl Agar Distilled water PH

Gram/Liter 14.0 4.5 4.5 5.0 15.0 1000ml 7.3

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

KF Streptococcal (KFSA) Agar

Gram/Liter

Protease Peptone Yeast Extract NaCl Sodium Glycerophosphate Maltose Lactose Sodium Azide Brom cresol purple Agar PH

10.0 10.0 5.0 10.0 20.0 1.0 0.4 0.015 20.0 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Trepton- Sodium (T1-N1) Broth Trypticase NaCl Distilled water PH

Gram/Liter 10.0 10.0 1000ml 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes Mueller Hinton Agar Beef infusion Bacto casamino acid (technical) Starch Bacto agar Distilled water PH

Gram/Liter 2.0 17.5 1.5 17.5 1000ml 7.3

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Mueller Hinton Broth Beef, infusion from Casein Hydrolysate (Acid) Soluble Starch PH

Gram/Liter 300 17.5 1.5 7.4

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Sodium Chloride Broth

Gram/Liter

Beef extract NaCl Distilled water

0.1 0.375 1000ml

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Endo Agar

Gram/Liter

Peptone Lactose Dipotasssum phosphate Sodium sulphat Basic Fuchsin Agar PH

10.0 10.0 3.5 2.5 0.5 15.5 7.5

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Mannitol Salt Agar

Gram/Liter

Proteas peptone Beef extract D-Mannitol NaCl Phenol red Agar Distilled water

10.0 1.0 10.0 75.0 0.025 20 1000ml

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Normal Saline NaCl Distilled water

Gram/Liter 0.85 1000ml

Autoclaved at 1210C for 15 minutes.

APPENDIX-II Composition of the media used in biochemical test MR-VP broth Peptone Dextrose Dipotassium phosphate Distilled water PH

Gram/Liter 7.0 5.0 5.0 1000ml 6.9

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Triple Sugar Iron (TSI) Agar

Gram /Liter

Peptone Tryptone Yeast Extract Lactose Saccharose Dextrose Ferrous Sulphate Sodium Chloride Sodium Thiosulphate Phenol Red Agar PH 7.4 Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Simmons citrate Agar Magnesium sulphate Manoammonium phosphate Dipotassium phosphate Sodium citrate Sodium Chloride Agar Brom-Thymol Blue PH

10.0 10.0 3.0 10.0 10.0 1.0 0.2 5.0 0.3 0.024 12.0

Gram/Liter 0.2 1.0 1.0 2.0 5.0 15.0 0.08 6.8

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Urea broth medium Urea Yeast extract KH2PO4

Gram/Liter 20.0 0.1 9.0

K2HPO4 Phenol red Distilled water PH Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Indol tryptopon broth medium

9.5 0.01 1000ml 6.8

Gram/Liter

Tryptone Distilled water

10.0 1000ml

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Carbohydrate fermentation media

Gram/Liter

Mannitol Beef extract Peptone NaCl Bromocresol purple Distilled water PH

l5.0 1.0 10.0 5.0 0.015 1000 6.8

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

Nitrate broth

Gram/Liter

Peptone Beef extract NaCl Potassium nitrate Agar Distilled water PH

5.0 3.0 5.0 1.0 1.0 1000ml 7.2

Sterilized at 1210C under 151b/in2 pressure for 15 minutes.

APPENDIX-III Composition of chemicals and reagents Crystal violet Solution-A Crystal violet (90% dye content) Ethyl alcohol (95%) Solution-B Ammonium oxalate Distilled water

2.0g 20.0ml 0.8 80.0 ml

Note-Mix the solution A and B Gram’s iodine Iodine Potassium iodide Distilled water

1.0g 2.0g 300.0ml

Ethyl alcohol (95%) Ethyl alcohol (100%) Distilled water

95.5ml 5.0ml

Safranin Safranin O Ethyl alcohol (95%) Distilled water

0.25ml 10.0ml 100.0ml

Methyl red solution Methyl red .04g Ethanol 40g Distilled water 100ml Methyl red dissolved in ethanol and diluted water.

Malachite green Malachite green Distilled water r

5.0g 100ml

Kovac’s reagent (for detection of indole) P-Dimethylaminobenzaldehyde Amyl alcohol Hydrochloric acid (concentrated)

5.0g 75.0ml 25.0ml

concentrated P-Dimethylaminobenzaldehyde was dissolved in the amyl alcohol and HCl was added slowly.

Barrit’s reagent Solution-A α-naptho Ethanol(Absolute)

l5.0g 95.0g

α-naptho was dissolved in ethanol with constant stirring.

Solution-B

KOH Creatine Distilled water

40.0g 0.3g 100ml

Hydrogen peroxide 3% aqueous solution of H2O2 was prepared from the H2O2 absolute solution.