INTRODUCTION:

Sewage waste water consists of a large number of pathogenic bacteria that remains either in single form or in clusters. Enteric bacteria are the most prevailing microbes present in the sewage waste water and soils. Different species of Salmonella have been reported by various researches¹.

Soil microbes play an important role in biogeochemical cycles. The microorganisms residing in soil influence the ecosystem by providing nutrition to plants.

Soil bacteria are actively involved in carbon fixation, decomposition of organic matter, respiration, nitrogen fixation, nitrogen mineralization, nitrification, denitrification, phosphorus mineralization and sulfur oxidation-reduction. These factors again directly or indirectly affect the plant and animal habitats ². Among the wide range of bacteria present in soil, only 1% can be isolated by laboratory techniques³.

The bacterial genus present in soil can be listed as Agrobacterium, Alcaligenes, Arthrobacter, Bacillus, Flavobacterium, Micromonospora, Nocardia, Pseudomonas, and Streptomyces⁴. However, upon contamination of the natural soil with some pollutants, the microbial habitat changes and pathogenic bacteria begin to endure in the contaminated soil. Contaminations of soil are mostly observed due to hospital waste water, industrial waste water and household waste water combining to the soil and thus enhancing pathogenic bacterial growth. Chemical contaminants include organic particles (faeces, hairs, food, vomit, paper fibres, plant material, humus, etc.), soluble organics (urea, fruit sugars, soluble proteins, drugs, pharmaceuticals, etc.), inorganics (sand, grit, metal particles, ceramics, etc.), soluble inorganics (ammonia, road-salt, sea-salt, cyanide, hydrogen sulfide, thiocyanates, thiosulfates, etc.), animals (protozoa, insects, arthropods, small fish, etc.), macro-solids (sanitary napkins, nappies/diapers, condoms, needles, children's toys, dead animals or plants, body parts, etc.), gases (hydrogen sulfide, carbon dioxide, methane, etc.) and toxins (pesticides, poisons, herbicides, etc.). These contaminants along with sewage water mixes with the soil and results in contaminated soil⁵. Soil contaminated with hospital sewage water mostly consists of bacteria like Escherichia coli, Citrobacter and Salmonella⁶. These strains are often resistant to most of antibiotics. The multi-resistant pathogenic strains enter the food chain once hospital sewage water or the soil is used for agricultural purposes⁷.

In this experiment different soil samples were collected from various sewage areas and bacteria were isolated from the soils. Biochemical tests of the bacterial isolates were performed with the isolates for identification of the bacteria. The bacterial isolates were inoculated on selective media and antibiotic sensitivity assay was performed to determine the sensitivity or resistance of the predominant bacteria against various antibiotics.

MATERIALS AND METHODOLOGY:

Collection of Sewage Soil Sample:

Soil was collected from 5 different sewage sites in Gandhinagar, Katpadi, Vellore, Tamil Nadu.

Treatment of the Soil Samples:

The soils were sun dried and sieved thoroughly with 2mm size sieve before proceeding with the experiment.

Isolation of Bacteria from the Sewage Soil Samples:

Nutrient agar was used for bacterial isolation from the sewage soil samples. 1 g of each soil sample was added to test tubes containing 10 ml of sterile distilled water for preparation of stock solution. Dilutions were prepared from each stock solution till 10^-3. 1 ml aliquot from each dilution was spread uniformly on nutrient agar plates and incubated for 24 h to check for bacterial growth.

Gram Staining of the Bacterial Isolates:

The isolated bacterial strains were stained with Gram staining procedure to detect the Gram characteristics of the strains. Gram positive bacteria are can be observed as purple whereas, Gram negative bacteria have pinkish appearance when observed under microscope ⁸.

Morphological Characterization of the Isolates:

The shapes of bacterial isolates were viewed under microscope during Gram staining.

Biochemical Tests:

Biochemical tests were performed to screen the bacterial isolates. IMViC test (Indole, Methyl red, Voges Proskauer and Citrate Utilization), triple sugar iron, urease, catalase, oxidase, nitrate reductase and carbohydrate fermentation test were conducted for all the bacteria isolated from the sewage soil sample.

IMViC Test:

IMViC test was performed to detect the coliform bacteria and the characteristics of them. IMViC test includes a series of four tests including indole, methyl red, Voges Proskauer and citrate utilization test.

Indole test was conducted to detect the ability of the isolated bacteria to produce indole after degradation of tryptophan. Tryptophan peptone broth was prepared, sterilized and inoculated with the isolated bacteria. One test tube with the broth was kept as control. After 24-48 h of incubation, 1 ml of KOVAC’s reagent was gently added to the bacterial culture and the control tubes. Observance of cherry red ring in the test tubes was considered as indole positive⁹.

Methyl Red- Voges Proskauer (MR-VP) test was performed with the bacterial isolates. MR-VP broth was prepared and inoculated after sterilization. Two sets of test tubes with MR-VP broth were maintained for methyl red and Voges Proskauer test individually. The test tubes with bacterial inoculated media were incubated for 48-72 h. After bacterial growth was observed, methyl red solution was added drop-wise to one set of bacterial isolates including a control and checked for colour change. Positive test is indicated by change of the broth to red colour, however, turning the colour to yellow depicts a negative result¹⁰. Voges Proskauer test was further performed with the other set of test tubes containing the isolated bacterial cultures. Alpha-naphthol and potassium hydroxide were added dropwise one after another and observed for colour change. The change of colour to red confirms the bacteria to be VP positive ¹¹.

The bacterial isolates were then checked for citrate utilization test. Simmon’s citrate agar was prepared for this test. The Simmon’s citrate agar contains citrate as its carbon source and bromothymol blue acts as the indicator. The agar was sterilized and inoculated with the bacterial isolates to observe the colour change after 3 days. The change of colour to blue demonstrates the bacteria to be positive for citrate utilization ¹².

Triple Sugar Iron Test:

Triple Sugar Iron test categorizes between Enterobacteriaceae and other members of Gram negative bacteria. Triple sugar iron (TSI) agar slants were prepared and inoculated with the bacterial isolates. After 24-48 h of incubation, the slants were observed for metabolization of sugars along with or without acid, gas and hydrogen sulphide production¹³.

Urease Test:

Urea agar was used for urease test. Phenol red was utilized as indicator for the test. The agar slants were prepared and sterilized followed by inoculation of bacterial isolates. The test tubes were incubated for 24-48 h at 37°C and observed for the colour change. The change in colour of the slants from yellow to red indicated that the bacteria to be urease positive¹⁴.

Catalase Test:

One loopfull of the isolated bacterial cultures were taken on the slide and 3% hydrogen peroxide was added drop-wise to observe the bubble formation. The formation of bubbles concluded the bacteria to be catalase positive.

Oxidase Test:

The test included formation of smear on a filter paper soaked in tetramethyl-p-phenylenediamine dihydrochloride and observed for a colour change to deep blue. The change of the inoculated filter paper to deep blue colour confirms the bacteria to be oxidase positive¹⁵.

Nitrate Reductase Test:

The nitrate test was conducted to detect nitrate production by the bacterial isolates. The test was performed to determine the ability of the bacterial isolates to reduce nitrate to nitrite. Nitrate broth was prepared, sterilized and inoculated with bacterial isolates. After 24-48 h of incubation, drops of sulfanilic acid and alpha-naphthylamine were added to the cultures. The change of the colour of broth to red indicated the presence of nitrite in the broth. However, no colour change proved as a negative result.

Carbohydrate Fermentation Test:

Carbohydrate fermentation broth was performed to check the carbohydrate fermentation capability of the isolated bacteria. Phenol red carbohydrate fermentation media was used during this test. Phenol red served as the indicator in carbohydrate fermentation test. Six different carbohydrate sources (glucose, sucrose, lactose, manitol, arabinose and rhamnose) were used for the test. Carbohydrate fermentation broth was prepared with varying concentration of different carbohydrates. Durham’s tubes were inserted in the test tube to observe the acid or gas production by the bacterial isolates. Once Durham’s tubes were inserted and sterilization of media was over, the bacteria were inoculated in the broth and incubated for 24-48 h to observe the colour change.

Identification of Predominant Strains:

After successfully completion of all these above explained tests, the bacterial isolates were identified based on their morphology and biochemical characteristics. Seven predominant strains were selected out of all the seventeen bacteria isolated and were further confirmed by the following experiments. The predominant strains identified were Escherichia coli, Salmonella, Shigella, Pseudomonas, Klebsiella, Staphylococcus and Streptococcus.

Growth of the Bacterial Isolates on Selective Media:

The presence of the seven predominant bacterial strains was confirmed by their growth on selective media. The selective media were chosen based on the identified bacteria. Blood agar and Mac Conkey agar were used for three isolates, such as, Escherichia coli, Salmonella and Shigella. Blood agar was also used to check the Streptococcus growth. However, for Escherichia coli Eosin methylene blue (EMB) agar was chosen and for Salmonella and Shigella, Salmonella Shigella agar medium was used. Mac Conkey agar was used for confirmation of the Pseudomonas and Klebsiella growth. The Staphylococcus isolation was confirmed by culturing them on Manitol Salt agar.

Antibiotic Sensitivity Assay:

Once the isolation of the bacterial strains was confirmed, the sensitivity of the bacterial strains against antibiotics was determined. The bacterial cultures were swabbed on Mueller Hinton agar plates. Antibiotic discs were then placed on the plates and incubated for 24-48 h to observe zone of clearance.

RESULTS AND DISCUSSIONS:

Isolation of Bacteria:

Seventeen bacterial isolates were isolated from the sewage soil samples. The biochemical tests were conducted for identification of the isolates.

Gram Staining:

The bacteria were stained following Gram staining procedure where they were differentiated based on their cell wall characteristics. The isolates were classified as Gram positive and Gram negative. The primary stain used for Gram Staining, crystal violet, in this case, are taken up by both the Gram positive and Gram negative cell walls based on the reaction of the acidic groups of the cell with the basic groups of the stain. The Gram’s iodine added during the staining procedure acts as a mordant which helps to bind the primary stain to the bacterial cell. In the third step, the slide with smear was dipped in alcohol to dissolve the dye-iodine complex from the Gram negative cell wall which results in colorless Gram negative cells. The Gram negative cells were then coloured by adding the counterstain, that is, safranin. Hence, after completion of the gram staining procedure, Gram positive cells were observed in purple whereas pink colour cells were identified as Gram negative bacteria ¹⁶. (Table-1)

Morphology Characterization:

The shapes of the bacterial isolates were observed during Gram staining observation at 100X under microscope. The Gram positive cells were cocci-shaped and the Gram negative cells were rod-shaped in morphology. The shapes are listed below in table 2.

Biochemical Tests:

The biochemical test for bacterial isolates performed included IMViC, triple sugar iron test, urease, catalase, oxidase and nitrate reduction tests. Indole test, methyl red, Voges Proskauer and Citrate Utilization test completed the IMViC test. IMViC test along with the other above mentioned tests aids in identification of Enterobacteriaceae¹⁷. The results obtained after completion of the biochemical tests were compared to the standard biochemical chart for preliminary identification of the bacterial isolates.

Table 1: Gram staining characteristics of the bacteria isolated from sewage soil sample

Isolates	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
Gram +/-	-	-	-	-	+	-	-	-	-	-	-	-	-	-	+	+	+	+

Table 2: The shapes of the bacteria as observed under 100X (R: rod, C: Coccus)

Isolates	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
Shape	R	R	R	R	C	R	R	R	R	R	R	R	R	R	R	C	C	C

Table 3: The biochemical analysis of the several isolates (I: Indole, MR: Methyl Red, VP: Voges Proskauer, C: Citrate Utilization, TSI: Triple Sugar Iron, U: Urease, CA: Catalase, O: Oxidase, N: Nitrate test)

Isolates	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
I	+	-	+	+	-	-	+	+	-	+	+	-	-	+	-	-	-	-
MR	+	+	+	+	+	+	+	+	-	-	+	-	-	+	-	+	+	+
VP	-	-	-	-	-	-	-	-	+	+	-	+	-	-	-	-	-	-
C	-	+	+	-	-	+	+	-	+	+	+	+	+	-	-	-	-	-
TSI	-	+	-	+	-	+	+	-	-	-	-	+	+	+	-	+	+	+
U	+	-	-	+	+	-	-	-	+	+	-	+	-	+	-	-	+	+
CA	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	-	-	-
O	-	-	-	-	-	-	+	-	-	+	-	-	+	-	+	-	-	-
N	-	+	-	+	-	-	-	-	+	+	+	+	+	-	+	-	-	-

Table 4: Results of Carbohydrate Fermentation Test with the isolated bacterial cultures

Isolates	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
Glucose	+	+	+	+	+	+	+	+	+	+	+	+	+	+	-	+	+	+
Sucrose	+	+	+	+	+	+	+	-	+	-	-	+	-	+	+	+	+	+
Lactose	+	-	+	-	-	-	+	-	+	-	-	+	-	+	-	-	-	-
Manitol	+	+	+	-	+	+	+	+	+	+	+	+	+	+	+	+	-	-
Arabinose	+	+	-	-	-	+	+	-	+	-	-	+	-	+	-	-	-	-
Rhamnose	-	+	-	-	-	+	+	-	+	-	-	+	-	-	+	-	-	-

Table 5: Identification of bacterial isolates based on the Gram Staining and Biochemical activity

Isolates	Name of the Identified Bacteria
1	Escherichia coli strain 1
2	Salmonella sp. strain 1
3	Shigella sp. strain 1
4	Proteus mirabilis
5	Staphylococcus aureus strain 1
6	Salmonella sp. strain 2
7	Citrobacter sp. strain 1
8	Shigella sp. strain 2
9	Klebsiella pneumonia strain 1
10	Pseudomonas aeruginosa strain 1
11	Shigella sp. strain 3
12	Klebsiella pneumonia strain 2
13	Pseudomonas aeruginosa strain 2
14	Escherichia coli strain 2
15	Serratia marcescens
16	Staphylococcus aureus strain 2
17	Streptococcus sp. strain 1
18	Streptococcus sp. strain 2

Carbohydrate Fermentation Test:

The ability of the bacterial isolates to ferment glucose, sucrose, lactose, manitol, arabinose and rhamnose was detected. The production of acid and gas by the bacterial strains were noted and considered during identification of the isolated bacterial strains.

Identification of Predominant Bacteria:

After conducting all the previous studies and comparing the results, the bacterial isolates were identified and the predominant isolates were chosen for the following experiment.

The Gram staining and biochemical activities showed that the predominant bacterial strains present in the sewage soil samples. Escherichia coli, Salmonella, Shigella, Pseudomonas, Staphylococcus, Streptococcus and Klebsiella were chosen for further experiment to confirm the presence of the above mentioned bacteria in the soil.

Growth of the Bacteria on Selective Media:

Screening of Escherichia Coli on Selective Media

Escherichia coli were screened by inoculation of isolate in Mac Conkey and Eosin methylene blue agar. Eosin methylene blue acts as both differential and selective media. The media enhances the growth of Gram negative bacteria while inhibiting the Gram positive bacterial totally. Lactose fermenter bacteria are able to grow on the EMB agar plates. Hence, the isolate grown of the eosin methylene blue agar plates were confirmed as Escherichia coli. However, growth of isolate on Mac Conkey agar and Blood agar supported the result. The isolated E. coli showed beta hemolysis when cultured on blood agar.

(a)

(b)

(c)

Figure 1: Growth of E. coli on (a) EMB, (b) Mac Conkey and (c) Blood agar

Screening of Salmonella on Selective Media

Salmonella growth was checked on Salmonella Shigella agar, Blood agar and Mac Conkey agar. The isolate showed black centered colonies when grown on Salmonella Shigella agar. However, no hemolysis on blood agar confirms the isolate to be Salmonella.

(a)

(b)

(c)

Figure 2: Growth of Salmonella on (a) Salmonella Shigella, (b) blood and (c) Mac Conkey agar

Screening of Shigella on Selective and Differential media:

Shigella, being non-lactose fermenting bacteria, are known for producing transparent colonies on Mac Conkey agar. The isolated bacteria produced transparent colonies on Mac Conkey agar which indicates the bacterial isolates to be Shigella. The result was however confirmed by transparent colonies on Salmonella Shigella agar and no hemolysis on blood agar.

(a)

(b)

(c)

Figure 3: Growth of Shigella on (a) Salmonella Shigella, (b) blood and (c) Mac Conkey agar

Screening of Pseudomonas on Selective Media:

Pseudomonas produced colorless colonies on Mac Conkey agar. Pseudomonas is non-lactose fermenting bacteria and the presence of colorless transparent colonies was observed.

Screening of Staphylococcus on Selective Media:

Manitol Salt agar acts as both differential and selective media for detecting the growth of Staphylococcus. The colonies appeared yellow colored and also showed light yellow zones formed around the colonies which indicated the utilization of Manitol by the organism. Thus the isolated bacteria were confirmed to be Staphylococcus.

Screening of Streptococcus on Selective Media:

The growth of Streptococcus was confirmed by observing beta hemolysis on the blood agar. The isolated bacteria was able to break the red blood cells completely and thus produced a clear zone around the colonies which indicated the isolated organism to be Streptococcus.

Screening of Klebsiella on Selective Media:

The isolated strain showed convex and mucoid colonies. Klebsiella, being a lactose positive strain produces mucoid, convex colonies. The colonies observed on the Mac Conkey agar indicated the organism to be Klebsiella.

Antibiotic Sensitivity Assay of the Bacterial Strains:

All the seven isolates, that is, Escherichia coli, Salmonella Shigella, Pseudomonas, Staphylococcus, Streptococcus and Klebsiella were resistant to penicillin, ampicillin and ceftazidime. Therefore, it can be concluded that the organisms showed resistance towards beta-lactam drugs.

Table 6: The effect of the bacterial strains against antibiotics (Ch: Chloramphenicol, C: Ciprofloxacin, T: Tetracycline, O: Ofloxacin, P: Penicillin, S: Streptomycin, A: Ampicillin, Ce: Ceftazidime) (R: resistant, S: Sensitive)

Sl. No.

Drugs

Disc Concentration

Name of the Bacteria

E. coli

Salmonella

Shigella

Pseudomonas

Staphylococcus

Streptococcus

Zone of Inhibition

Interpretation

Zone of Inhibition

Interpretation

Zone of Inhibition

Interpretation

Zone of Inhibition

Interpretation

Zone of Inhibition

Interpretation

Zone of Inhibition

Interpretation

30µg

10µg

5 µg

10 units

10µg

30 µg

Figure 4: The antibiotic sensitivity assay indicates that the bacterial strains are resistant to penicillin, ampicillin and ceftazidime.

CONCLUSION:

Soil samples were collected from five different sewage areas. The predominant presence of Escherichia coli, Salmonella, Shigella, Pseudomonas, Staphylococcus, Streptococcus and Klebsiella in the soil samples was identified. Antibiotic sensitivity assay was performed with the predominant bacterial strains. The isolates showed resistance to beta lactam drugs, such as, penicillin, ampicillin and ceftazidime. Hence, the bacterial isolates were confirmed to be Escherichia coli, Salmonella, Shigella, Pseudomonas, Staphylococcus, Streptococcus and Klebsiella.

ACKNOWLEDGEMENT:

The authors are greatly indebted to Vellore Institute of Technology, Vellore for their constant encouragement, help and support for extending necessary facilities to successfully complete the experiment.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

REFERENCES:

1. Teltsch B, Kedmi S, Bonnet L, Borenzstajn-Rotem Y, Katzenelson E. Isolation and identification of pathogenic microorganisms at wastewater-irrigated fields: ratios in air and wastewater. Applied and Environmental Microbiology. 1980 Jun 1; 39(6):1183-90.

2. Sides KE. Agricultural Soil Bacteria; A Study of Collection, Cultivation, and Lysogeny. 2010.

3. Nandhini B and Josephine RM. Original Research Article A study on bacterial and fungal diversity in potted soil. Int. J. Curr. Microbiol. App. Sci. 2013; 2(2):1-5.

4. Janssen PH. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Applied and environmental microbiology. 2006 Mar 1; 72(3):1719-28.

5. El Bestawy E, Ahmed AH, Amer R, Kashmeri RA. Decontamination of Domestic Wastewater Using Suspended Individual and Mixed Bacteria in Batch System. Journal of Bioremediation and Biodegradation. 2014; 2014.

6. Moges F, Endris M, Belyhun Y, Worku W. Isolation and characterization of multiple drug resistance bacterial pathogens from waste water in hospital and non-hospital environments, Northwest Ethiopia. BMC research notes. 2014 Apr 5; 7(1):1.

7. Ibrahim MK, Galal AM, Al-Turk IM, Al-Zhrany KD. Antibiotic resistance in Gram-negative pathogenic bacteria in hospitals' drain in Al-Madina Al-Munnawara. Journal of Taibah University for Science. 2010 Dec 31; 3:14-22.

8. Selladurai G, Anbusaravanan N, Teixeira da Silva JA, Shyam KP, Kadalmani B. Dynamics of microbial diversity, micro-and macronutrients during tannery sludge treatment using epigeic earthworms, Eisenia fetida and Eudrilus eugeniae. Vermitechnology II. Dynamic Soil, Dynamic Plant. 2010; 4:100-7.

9. Vashist H, Sharma D, Gupta A. A review on commonly used biochemical test for bacteria. Innovare Journal of Life Sciences. 2013 May 20; 1(1):1-7.

10. Cowan ST. Micromethod for the Methyl red test. Microbiology. 1953 Aug 1; 9(1):101-9.

11. Barry AL and Feeney KL. Two quick methods for Voges-Proskauer test. Applied microbiology. 1967 Sep 1;15(5):1138-41.

12. Siddiquie MD and Mishra RP. Age and Gender wise Distribution Pattern of Typhoid causing Bacteria Salmonella serovars in Mahakaushal Region. World Journal of Pharmaceutical Research. 2014; 3(4):1183-1203.

13. Ateba TP, Moroane T, Nyirenda M, Gopane RE, Ateba CN. Detection of antibiotic resistant Enterobacteriaceae from dogs in North West University (South Africa) animal health hospital. African Journal of Microbiology Research. 2013 Oct 25; 7(43):5004-10.

14. Holding AJ, Collee JG. Chapter I Routine Biochemical Tests. Methods in microbiology. 1971 Dec 31; 6:1-32.

15. Steel KJ. The oxidase reaction as a taxonomic tool. Microbiology. 1961 Jun 1; 25(2):297-306.

16. Bartholomew JW and Mittwer T. The gram stain. Bacteriological reviews, 1952; 16(1): p.1.

17. Varkey DR, Balaji V, Abraham JA. Molecular Characterisation of Extended Spectrum Beta Lactamase producing strains from blood sample. Int J Pharm Pharm Sci. 2014;6:276-8.

Received on 19.01.2017 Modified on 25.03.2017

Research J. Pharm. and Tech. 2017; 10(4): 1053-1059.

DOI: 10.5958/0974-360X.2017.00191.3