INTRODUCTION:

Hospitals and health-care centres are considered to be one of the prime consumers of water. Water is used for different purposes like drinking, cleaning and for other broad-spectrum therapeutic applications making it an integral part of any health-care unit. As a result, these establishments generate substantial amount of wastewater with comparable quality to that of metropolitan sewage. The hospital discharges contain different potentially toxic and hazardous clinical substances, both organic and inorganic in nature like lethal chemicals (cleaning and disinfection operation), pathogenic microbes (enteric pathogens transmitted via water from infected patients with a particular enteric disease), drug metabolites (antibiotics and genotoxic medications in the form of micropollutants), suspended solids, heavy metals and radioactive isotope (wastes from oncology departments)^1-7.

Hospital sewage is a common habitation for a large number of pathogenic microbes with characteristics like drug and heat resistance, heavy metal tolerance etc.

which vary slightly in nature when compared to the other sewages (municipal and industrial). Literature review indicated the presence of pathogenic strains of bacteria which were usually found to be present in a consortium of different cell types rather than in its single state. These bacterial strains showed the resistance towards multiple drugs presenting an all new level of threatening contamination^3,4. Soil contaminated with these pathogenic strains (from hospital sewage) enters the food web if they are used for agricultural purposes^8,9.

The most common pathogens to be found in the sewage water and soil are possibly the enteric bacteria, of which Salmonella sp., Citrobacter sp. and E.coli are found to be prevailing more frequently by maintaining their viability in these sites^10,11. Some researchers have suggested that these enteric bacteria can be used as an indicator of water contaminations whereas other group of researchers have emphasized using coliform bacteria as a universal indicator of soil contaminations caused by sewage outflow from the hospitals^12-14. Upon soil contamination, these microorganisms directly or indirectly affect the flora and fauna¹⁵. They play a vital role in the decomposition of organic matter thereby releasing the nutrients into the soil in its mineral form which can be later taken up by the plants for their vital processes like photosynthesis, growth and development. Apart from these, the soil microorganism’s function in carbon and nitrogen fixation, oxidation reduction of iron, ammonia and sulphur, phosphorous mineralization etc. despite the diversity in soil microbiology, less than 1% of the microorganisms can be cultivated in the laboratory¹⁶. The bacterial genera predominantly existing in the soil are taxonomically comprised of three orders, Pseudomonadales, Eubacteriales and Actinomycetales. Under these, the most common soil bacteria belong to the genera Shigella, Achromobacter, Klebsiella, Pseudomonas, Polyangium, Flavobacterium, Arthrobacter, Clostridium, Bacillus, Sarcina, Enterobacter, Nocardia, Chondrococcus, Micrococcus, Archangium, Cyptophaga, Agrobacterium etc¹⁷.

The following investigation was conducted to analyse the soil microbiology of samples collected from hospital sewage followed by the biochemical characterization of the bacterial isolates for its primary identification. Antibiotic resistance assay was performed to determine the sensitivity or resistance of the bacterial isolates against different antibiotics.

MATERIALS AND METHODOLOGY:

Chemicals:

All the media, chemicals and antibiotic discs used in the following study was purchased from Hi Media Laboratories, Mumbai, India and Sisco Research Laboratories (SRL) Private Limited, Mumbai, India.

Collection of Sewage Soil Samples:

Soil samples were collected from hospital sewage in and around Vellore, Tamil Nadu, India.

Pre-treatment of the Soil Samples:

Preceding the experimental procedures, the soil samples were sun dried for 24 hours in order to reduce the number of fungal colonies. Post drying, the samples were sieved through 2 mm sized mesh to separate granules and pebbles.

Bacteria Isolation from the sewage samples:

Isolation and enumeration of bacterial colonies from dried sewage samples were performed using the serial dilution method. Stock solution of the sample was prepared by adding 1 gram of the dried soil sample to sterile distilled water which was serially diluted. Dilutions ranging from 10^-3 to 10^-5was evenly spread over sterilized Nutrient Agar plates. The plates were incubated and checked for bacterial growth after 24 hours. The suspected colonies were sub cultured and stored at 4ºC until further use.

Gram Staining of the Isolates:

The isolated bacterial strains were subjected to Gram staining in order to determine their respective Gram’s characteristics. Gram positive bacteria were observed as purple colored cells while Gram negative bacteria were pink in color when observed under light microscope of varying magnification¹⁸. The shapes of the bacterial cells were observed under microscope during Gram staining technique.

Morphological Characteristics of the Isolates:

The morphology, such as the shape, color, size and structure of the bacterial colonies were observed on the nutrient agar plate.

Biochemical characterization of the isolates:

The bacterial isolates were further subjected to their biochemical characterization. The following tests were performed: IMViC (Indole, Methyl red, Voges Proskauer and Citrate Utilization), urease, triple sugar iron, catalase, oxidase, nitrate reductase test and carbohydrate fermentation test.

IMViC Test:

This biochemical assay is a general test consisting of four separate reaction carried out individually. The tests are Indole production, methyl Red test, Voges Proskauer test and citrate utilization¹⁹.

Indole production:

The isolates are subjected to the indole production test in order to check for the ability of the organism to degrade tryptophan present in the tryptophan peptone broth via enzyme tryptophanse along with the production of indole molecule as one of the end product. The media was inoculated with the test isolates and incubated for 24 hours. Control tubes were maintained. One ml of Kovac’s reagent is added to the culture broth to check for the production of indole. The positive tube was confirmed by the formation of cherry red/ dark pink colour ring on the top layer of the culture broth.

Methyl Red- Voges Proskauer (MR-VP) test:

The following biochemical test was performed individually in two different sets of MR-VP broth. Sterilised broth was inoculated and incubated for 24-72 hours. Control tubes were maintained. A drop of methyl red was added to one set of the tube and observed for the change in the broth colour. Positive test was indicated by the formation of red colour while yellow colour was taken as the negative test²⁰. Voges Proskauer biochemical test was performed with the other set of culture tubes containing the bacterial isolates. To the tubes, α-naphthol and potassium hydroxide was added and observed for colour change. Positive test was indicated by the formation of red/ pinkish-red colour due to the presence of acetoin²¹.

Citrate utilization test:

The bacterial isolates were further screened for their ability to utilise citrate as their sole carbon source. The isolates were inoculated in sterilised Simmon’s citrate agar containing citrate as the sole carbon source and bromothymol blue as an indicator. Control tubes were maintained .The inoculated tubes were incubated for 3 days and observed for change in colour. The appearance of red colour post the incubation time, indicated positive result²².

Triple Sugar Iron (TSI) test:

The bacterial isolates were checked for their ability to ferment various sugars with the production of hydrogen sulphide, acids or gas, which are pH sensitive. It is a differential test used to differentiate the enteric bacteria based on their fermentation ability. Sterilized TSI slants were prepared and inoculated with the test isolates. This is followed by incubation for 24 hours. Control tubes were maintained. Post incubation, the culture tubes were observed for colour change. Red colour infers the fermentation of only glucose sugar, yellow colour infers the fermentation of both glucose and lactose sugar, black colour infers the production of hydrogen sulphide²³.

Urease test:

Urease test was performed to screen the bacterial isolates for its ability to hydrolyse urea into the by-products ammonia and carbon dioxide via the production of urease enzyme. Phenol red is present in the urea agar as an indicator. Sterilized agar slants were inoculated with the test bacterial isolates and further incubated for 24- 48 hours. Control tubes were maintained. Colour change was observed post the incubation time. The sign of yellow colour changing to red, indicated positive result²⁴.

Catalase test:

Catalase test was performed to check for the ability of the isolates to produce the catalase enzyme which could hydrolyse hydrogen peroxide into water and oxygen gas. A loopfull of the test isolates was taken on a clean glass slide. To the culture, 3% of hydrogen peroxide was added. Control tubes were maintained. Positive test was indicated by the formation of bubbles on the glass slide due to the production of oxygen gas form the reaction with hydrogen peroxide.

Oxidase test:

The test is used to check for the bacterial isolate that could produce the enzyme cytochrome c oxidase which is responsible for the electron transport chain system in them. To determine the presence of oxidase enzyme produced by the isolates, oxidase reaction was carried out. Each oxidase disk was touched and spread upon with a well isolated colony. Control disc was maintained. The reaction was observed within 5-10 seconds at 25-30˚C. A delayed positive reaction appears in 10-60 seconds while a change later than 60 seconds or no change at all is considered as a negative reaction. The formation of blue colour confirmed the test²⁵.

Nitrate reductase test:

The nitrate reductase test was performed to differentiate the bacterial isolates based on their ability to produce nitrate reductase enzyme which reduces nitrate to nitrite. Sterilized nitrate broth is inoculated with the test bacterial isolates and incubated for 24-48 hours. Control tubes were maintained. Post incubation period, sulphanilic acid and α-naphthylamine was added to the culture tube. Positive test is indicated by the change of colour of the culture broth to red.

Identification of the Isolated Bacteria:

The bacterial isolates were identified on the basis of the preliminary studies involving Gram staining, morphological studies and a series of biochemical assays.

Antibiotic Sensitivity Assay:

Post confirmations of the bacterial isolates up to the genus level, the isolates were subjected to antibiotic sensitivity assay. The isolates were cultured on sterilized Mueller Hinton agar plates. Sensitivity or resistance of the bacterial isolates against different antibiotics was determined. The following antibiotic discs were used in this assay- tetracycline, ofloxacin, ciprofloxacin, erythromycin, kanamycin, tigecycline, gentamycin, methicillin, linezolid, meropenem, pristinomycin, vancomycin, aztreonem, etrapenem, cefoxitin, clindamycin, imipenem, netillin, trimethoprim, chloramphenicol, cefotaxime and levofloxacin. The plates were incubated for 24- 48 hours. Post incubation, the zone of clearance was observed²⁶.

RESULTS AND DISCUSSIONS:

Isolation of Bacteria:

Five different bacteria were isolated from the hospital sewage samples which were identified after Gram staining, morphological studies and biochemical test.

Gram Staining and Morphological Study:

Gram staining of the isolated bacteria was done as the preliminary test to identify the Gram characteristics of the isolates. The Gram staining technique follows the principle of differentiating the bacteria based on the nature of their cell wall. The Gram positive bacteria were observed purple in colour due to the presence of thick peptidoglycan layer in them. The layer adhere the primary stain in it and after the binding activity with the help of mordant; it does not let the primary stain get washed away by the alcohol. On the other hand, a gram negative bacterial cell wall is composed of very thin layer of peptidoglycan and thus once dipped in alcohol the primary stain gets washed away which results in Gram negative cells showing the colour of the counter stain under microscope²⁷. The colonies formed on the nutrient agar plates were studied. It was observed that smooth surfaced colonies were produced.

Table 1: Gram Characteristics and Morphology analysis of Bacterial isolates

Sl. No.	Bacterial isolates	Gram +/-	Bacterial Shape	Colony Colour
1	SI	-	Rod	White
2	SII	+	Coccus	Yellow
3	SIII	-	Rod	White
4	SIV	-	Rod	White
5	SV	+	Coccus	White

Figure 1: Bacterial growth on Nutrient Agar plates

Figure 2: Gram Staining Observation of the Bacterial isolates

Biochemical characterization of the isolates:

After isolation and identification of the bacterial isolates via cultural methods and microscopic observation, the biochemical analysis for the said isolates was performed. The biochemical tests included the following: IMViC, Triple Sugar Iron test, Urease test, Catalase test, Oxidase test and Nitrate Reduction tests. IMViC test comprised of Indole test, Methyl Red test, Voges Proskauer test and Citrate Utilization test, aiding in identification and differentiation of Family Enterobacteriaceae upto genus level. The results obtained post the completion of all the biochemical tests were compared to the standard biochemical chart for preliminary identification of the bacterial isolates.

Table 2: Biochemical analysis of five bacterial isolates

Isolates	SI	SII	SIII	SIV	SV
Indole test	+	-	-	+	-
Methyl Red test	+	+	-	+	+
Voges Proskauer test	-	-	-	-	-
Citrate Utilization test	-	-	+	+	-
Triple Sugar Iron test	-	-	+	+	+
Urease test	+	+	-	-	+
Catalase test	+	+	+	+	-
Oxidase test	-	-	+	+	-
Nitrate Reductase test	-	-	+	-	-

Identification of the potential bacterial isolate:

Following the cultural, morphological and biochemical analysis, the results was compared. The predominant bacterial isolates were identified as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus sp.

Table 3: Identification of Bacterial Isolates from the hospital sewage soil sample

Sl. No.	Isolates Lab Code	Bacterial Isolates
1	SI	Escherichia coli
2	SII	Staphylococcus aureus
3	SIII	Pseudomonas aeruginosa
4	SIV	Klebsiella pneumoniae
5	SV	Streptococcus

Antibiotic Sensitivity Assay:

All the five isolates namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus sp. were resistant to kanamycin, gentamycin, methicillin, vancomycin, cefoxitin and netillin. Hence, it can be inferred that the bacterial isolates showed resistance against Aminoglycoside, narrow-spectrum β-lactam and cephalosporin whereas, the same isolates were sensitive against the action of the drugs namely erythromycin, linezolid, meropenem, and levofloxacin, which belongs to the class oxazolidinones and carbapenem.

Sl. No.	Name of the Antibiotic	Bacterial Isolates
		*E. coli*	*S. aureus*	*P. aeruginosa*	*K. pneumoniae*	*Streptococcus* sp.
1	Tetracycline	1.1	1.2	1.3	0.7	1.3
2	Ofloxacin	1.2	0.8	1	0.8	1.1
3	Ciprofloxacin	0.8	1.1	1	1	1.2
4	Erythromycin	1.3	1.2	1.2	1.1	1.3
5	Kanamycin	0.9	1	0.7	0.9	0.7
6	Tigecycline	1.1	0.2	0.4	0.8	0.6
7	Gentamycin	0.8	0.5	0.3	0.5	0..3
8	Methicillin	0.5	0.6	0.5	0.6	-
9	Linezolid	1.4	1.5	1	1.4	1.1
10	Meropenem	1.3	1.4	1	1.3	1
11	Pristinomycin	1	1.2	0.7	1	0.4
12	Vancomycin	0.6	0.6	0.3	0.5	0.5
13	Etrapenem	1.5	1.7	1.5	1.4	0.9
14	Aztreonem	0.5	0.3	0.6	1	0.7
15	Cefoxitin	0.8	-	0.9	0.9	0.3
16	Clindamycin	1	0.5	0.9	1	0.3
17	Imipenem	2	1.2	1.9	2.2	2
18	Netillin	0.6	0.4	0.6	0.7	0.6
19	Trimethoprim	1.5	0.7	1	0.3	1
20	Chloramphenicol	1	1.3	1.1	0.7	1.4
21	Cefotaxime	0.6	0.9	1	0.7	0.8
22	Levofloxacin	1.5	1.4	1.4	1.2	1

CONCLUSION:

Five bacterial strains were isolated from soil samples collected from hospital sewage areas of Vellore, Tamil Nadu. The bacterial strains obtained were Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus sp. The isolated bacteria were subjected to antibiotic sensitivity assay to interpret their multiple drug resistance activity. All the strains showed potent drug resistance against kanamycin, gentamycin, methicillin, vancomycin, cefoxitin and netillin.

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.

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Received on 26.06.2017 Modified on 11.07.2017

Research J. Pharm. and Tech 2018; 11(7): 2901-2905.

DOI: 10.5958/0974-360X.2018.00534.6