INTRODUCTION:

Use of sprouted seeds as food for human consumption originated in the Far East and because of their nutritive value, sprouted seeds have gained popularity worldwide. Sprouts are believed to be highly nutritious and rich in enzymes which promote good health. Sprouts, including mung beans and alfalfa sprouts, have become a common food item in grocery stores, salad bars around the world. Sprouting is the practice of germinating seeds to be eaten either raw or cooked. ^1,2,3 A large number of food borne disease outbreaks reported world over have been found to be linked to sprouts. In most instances, the illnesses were caused by either Escherichia coli O157:H7 or Salmonella bacteria.⁴

Thus it is necessary to control the contamination of seeds meant for consumption as sprouts or restrict the growth of pathogens during sprouting. The present study was undertaken to determine the antimicrobial susceptibility of isolated bacteria from moth bean sprouts.

MATERIAL AND METHODS⁴:

The present study was carried out in Department of Microbiology, Sardar Bhagwan Singh Post Graduate Institute of Biomedical Sciences and Research, Balawala, Dehradun.

Collection of Sample

Two seed samples of moth bean were purchased from local market from two different retail shops.

Seed Sprouting

Each type of seeds (5.0g) were placed in a sterile petri plate containing Whatmann No. 113 (qualitative wet strengthen) filter paper saturated with sterilized water or tap water. The seeds were incubated at room temperature (25^OC).

Microbial Analysis of Seed Sprouts

Sprouts after 24, 48, 72, 96 h of sprouting from 5.0 g of seeds were suspended in 95 ml sterile water blank. The flasks were placed on a rotary shaker for 15 minute and ten fold serial dilutions were prepared. Aliquots of 0.1 ml of appropriate dilutions were spread on plate containing Mac Conkey Agar, Brilliant Green Agar, Baired Parker’s agar and plates were incubated at 37^OC for 24 to 48 hrs.

The plates were observed after incubation for growth and colony Characteristics. Number of different colonies appearing on the plates was counted and number of bacteria present on each sprout was calculated as follows

No. of Bacteria per Sprout =

Number of colonies × dilution factor ×0.1

Number of seeds

Cultural Characterization

The colonies were observed for their color, texture, outline, opacity, pigmentation etc and different type of colonies appearing on all the three media were counted.

Morphological Characterization

The colonies were picked and processed for Gram staining technique to differentiate between Gram positive and Gram negative bacteria, shape and arrangement of cells.

Media Composition

Different types of media used in the study were Mac Conkey Agar, Brilliant Green Agar, Baired Parker’s agar etc., which were sterilized by autoclaving at121^OC for 20 minute at 15 lbs psi.

Mac Conkey Agar Medium (pH- 7.4)⁵
Ingredients		Amount (g/ l)
Peptone	:	5
Lactose	:	10
Bile salt	:	5
Sodium chloride	:	5
Neutral red	:	0.075
Agar	:	15
Brilliant Green Agar Medium (pH- 6.9) ⁶
Ingredients		Amount (g/l)
Lactose	:	10
Sucrose	:	10
Peptone	:	5
Sodium chloride	:	5
Phenol red	:	0.08
Brilliant green	:	0.0125
Agar	:	20
Baired Parker’s Medium (pH-7.0) ⁷
Ingredients		Amount (g/ l)
Casein enzyme hydrolysate	:	10
Beef extract	:	5
Yeast extract	:	1
Glycine	:	12
Sodium pyruvate	:	10
Lithium chloride	:	5
Agar	:	20
Muller Hinton Agar (pH 7.3)
Ingredients		Amount (g/ l)
Caesin acid hydrolysis	:	17.5
Beef heart infusion	:	2
Starch soluble	:	1.5
Agar	:	18

Drug Susceptibility (Intrinsic Antibiotic Resistance) Test

Due to emergence of many antibiotic resistant strains of bacteria, antimicrobial susceptibility testing is done in order to determine which antimicrobial agent to use against specific strain of bacteria. Antibiotic susceptibility was determined by disk diffusion method according to standard microbial procedures. In this method, a standard suspension of bacteria (0.5 turbidity Mc farland standard) to be tested was inoculated on the surface of Muller Hinton Agar plates. Filter paper disc containing specific concentration of antimicrobial agents were pressed onto the surface and incubated at 37^OC over night. After incubation, the zone of inhibition of growth of bacteria around each disc was measured and the susceptibility was determined.⁸

Disc of following antibacterial chemotherapeutic agents were used in this study: Ampicillin (10mcg/disc), Amoxycillin (30/15mcg/disc), Ciprofloxacin (10mcg/disc), Chloramphenicol (10mcg/disc), Oxacillin (1mcg/disc), Gentamicin (120mcg/disc), Voriconazole (1mcg/disc), Rifampicin (15mcg/disc) Methicillin (30 mcg/disc), Norfloxacin (10 mcg/disc).

RESULT AND DISCUSSION:

In the present study antibiotic susceptibility testing of bacterial isolates from moth bean sprouts was done. The observations of the experiments conducted were discussed as below:

Morphological Characteristics of Selected Isolates

List of isolates from different samples of moth bean sprouts picked at different time interval is given in table 1.

Drug Susceptibility (Intrinsic Antibiotic Resistance) of Selected Isolates

Antibiotic susceptibility of the isolates from moth bean sprouts was examined against 10 antibiotics viz Ampicillin (10mcg/disc), Amoxycillin (30/15mcg/disc), Ciprofloxacin (10mcg/disc), Chloramphenicol (10mcg/disc), Oxacillin (1mcg/disc), Gentamicin (120mcg/disc), Voriconazole (1mcg/disc), Rifampicin (15mcg/disc) Methicillin (30 mcg/disc), Norfloxacin (10 mcg/disc).

Antibiotic sensitivity pattern of Escherichia coli isolated from moth bean sprouts against 10 antibiotics were observed (Table 2). All the 5 isolates were found to be resistant to three antibiotics i.e Voriconazole, Methicillin and Ampicillin. Isolate number I-C2 and I-C3 were resistant to Rifamycin also since the zone of inhibition of these isolates was 9mm only. All other isolates were highly sensitive to the remaining antibiotics.

Antibiotic sensitivity pattern of Klebsiella sp. isolated from different sprouts is given in table 3. All these isolates were resistance to antibiotics like Voriconazole, Oxacillin, Amoxycillin. Isolate II-C1 was resistant to Rifamycin. Isolate II-C4, II-D2, and II-D3 were moderately sensitive to Rifamycin. All the isolates were highly sensitive to Chloramphenicol, Gentamycin, Voriconazol, Rifamycin and Norfloxacin.

Antibiotic sensitivity of 4 isolates of Salmonella sp. and 2 isolates of Shigella sp. isolated from moth bean sprouts on Brillant Green agar against 7 antibiotics is given in table 4 and 5. On the basis of size of zone of inhibition all the isolates of Salmonella sp. and Shigella sp. were found to be resistant to the Voriconazole and Oxacillin. Isolates viz. IV-C2, IV-C3 were moderately sensitive to Amoxicillin.

Antibiotic sensitivity pattern of Staphylococcus aureus isolated from moth bean sprouts was also studied. The observations are given in table 6. Eight isolates were tested and found to be resistant to Voriconzole, Methicillin and Oxacillin and were highly sensitive to the 5 remaining antibiotics namely Amoxycillin, Chloromphenicol, Amipicillin, Gentamycin and Ciprofloxacin.

Table 2: Antibiotic Sensitivity Pattern of E. coli isolated from Moth Bean Sprouts

Isolate no.	Antibiotics (Zone of Inhibition mm)
Isolate no.	Cf¹⁰	C¹⁰	AS¹⁰	G¹²⁰	VO R¹	Ox¹	Rf¹⁵	M³⁰
I-C2	38	23	_	27	_	27	9	_
I-C3	27	22	_	24	_	22	9	_
I-C4	29	27	_	28	_	30	12	_
I-D3	31	20	_	26	_	25	18	_
I-D4	26	25	_	22	_	23	16	_

G–Gentamicin , VOR –Voriconazole , C^_Chloromphenicol, Ox- Oxacillin, Cf - Ciprofloxacin, Rf -Rifampicin , A –Ampicillin, M-methicillin

Figures in superscript indicate concentration (mcg) of antibiotics.

Table 3: Antibiotic Sensitivity Pattern of Klebsiella isolated from Moth Bean Sprouts

Isolate no.	Antibiotics (Zone of Inhibition mm)
Isolate no.	Cf¹⁰	C¹⁰	AS¹⁰	G¹²⁰	VO R¹	Ox¹	Rf¹⁵	Nf¹⁰
II--C1	32	31	_	21	_	_	9	24
II-C2	34	26	_	18	_	_	14	21
II-C3	25	20	_	19	_	_	12	23
II-C4	27	21	_	21	_	_	12	25
II-D1	26	28	_	22	_	_	18	28
II-D2	30	32	_	27	_	_	14	27
II-D3	22	32	_	26	_	_	12	21
II-D4	23	27	_	23	_	_	10	24

G–Gentamicin, VOR-Voriconazole, C - Chloromphenicol, Cf - Ciprofloxacin, Rf - Rifampicin , A- Ampicillin, Nf- Norfloxacin, Ox- Oxacillin

Figures in superscript indicate concentration (mcg) of antibiotics

Table 4: Antibiotic Sensitivity Pattern Salmonella spp isolated from Moth Bean Sprouts

Isolate no.	Antibiotics Zone of inhibition (mm)
Isolate no.	CF¹⁰	C¹⁰	AS¹⁰	G¹²⁰	VO R¹	OX¹	AM S^30/15
IV-C1	31	21	24	23	_	_	21
IV-C2	30	24	22	22	_	_	15
IV-C3	29	20	28	27	_	_	14
IV-D2	34	25	21	21	_	_	16

G–Gentamicin ,VOR –Voriconazole , C-Chloromphenicol , Cf -Ciprofloxacin, A -Ampicillin, OX – Oxacillin, AMS –Amoxacillin

Figures in superscript indicate concentration (μg) of antibiotics

Table 5: Antibiotic Sensitivity Pattern Shigella isolated from Moth Bean Sprouts

Isolate no.	Antibiotics Zone of inhibition (mm)
Isolate no.	CF¹⁰	C¹⁰	AS¹⁰	G¹²⁰	VOR¹	OX¹	AM S^30/15
IV-C4	30	25	25	25	_	_	17
IV-D1	32	22	23	27	_	_	15

G–Gentamicin ,VOR –Voriconazole , C-Chloromphenicol , Cf - Ciprofloxacin, A -Ampicillin, OX – Oxacillin, AMS –Amoxacillin

Figures in superscript indicate concentration (μg) of antibiotics

Table 6: Antibiotic Sensitivity Pattern of Staphylococcus aureus isolated from Moth Bean Sprouts

Isolate no.	Antibiotics Zone of inhibition (mm)
Isolate no.	Cf¹⁰	C¹⁰	AS¹⁰	G¹²⁰	VOR¹	OX¹	AMS^30/15
V-C1	31	21	27	29	_	_	27
V-C2	31	24	23	26	_	_	30
V-C3	30	28	28	27	_	_	28
V-C4	32	29	25	24	_	_	32
V-D1	34	27	26	23	_	_	27
V-D2	31	23	24	25	_	_	29
V-D3	29	25	23	26	_	_	30
V-D4	33	21	28	28	_	_	31

G–Gentamicin, VOR–Voriconazole , C- Chloromphenicol, Cf - Ciprofloxacin, AS-Ampicillin, OX–Oxacillin, AMS –Amoxycillin

Figures in superscript indicate concentration (mcg) of antibiotics

SUMMERY AND CONCLUSION:

The present study was undertaken to determine the antimicrobial susceptibility of isolated bacteria from moth bean sprouts against 10 antibiotics viz. Ampicillin, Amoxycillin, Ciprofloxacin, Chloramphenicol, Oxacillin, Gentamicin, Voriconazole, Rifampicin, Methicillin, Norfloxacin.

Ø The isolates were picked from different samples on two types of media and purified for morphological characterization. They were found belonging to Escherichia coli, Klebsiella, Salmonella, Shigella and Staphylococcus aureus.

Ø The antibiotic susceptibility pattern of all the isolates was also determined. Isolates found from Escherichia coli were resistant to Ampicillin, Methicillin, and Voriconazole whereas isolates belonging to Klebsiella spp. were resistant to Ampicillin, Voriconazole, and Oxacillin.

Ø All the isolates of Salmonella spp. and Shigella spp. isolated on Brilliant Green Agar were resistant to Oxacillin and Voriconazole. Likewise all the isolates of Staphylococcus aureus isolated on Baired Parker’s medium were also resistant to Oxacillin, Methicillin and Voriconazole.

Ø Thus care should be taken in the consumption of the raw moth sprouts irrespective of their source i.e. whether prepared at home or procured from the retail market.

ACKNOWLEDGEMENT:

The Authors of this research articles are heartily thankful to their Guide and Head of Department of Microbiology, Sardar Bhagwan Singh Post Graduate Institute of Biomedical Sciences and Research, Balawala, Dehradun to provide all necessary help to construct such a fruitful work.

REFERENCE:

1. J. Augustin, B. P. Klein; Nutrient content of sprouted wheat and selected legumes, Cereal Foods World, 1983, 28, 58–361.

2. J. R. Fordham, C. E. Wells, L. H. Chen; Sprouting of seeds and nutrient composition of seeds and sprouts, J. Food Sci., 1975, 40, 552–556.

3. A. M. Kylen, R. M. McCready; Nutrients in seeds and sprouts of alfalfa, lentils, mung beans and soybeans, J. Food Sci, 1975, 40, 1008–1009.

4. Shilpi Chauhan, Ankit Saini, Devendra Pratap Singh, Girendra K. Gautam, Poonam Gupta. Antibiotic susceptibility of bacterial isolates from the sprouts of moth bean (Vigna Radiate L.), J. Pharm. and Biomed. Analysis Letters, 2013, 1(1), 40-44.

5. www.himedialabs.com/TD/M081B.pdf (Accessed on February, 2012).

6. www.himedialabs.com/TD/M016.pdf‎ (Accessed on February, 2012).

7. www.himedialabs.com/TD/M043.pdf (Accessed on February, 2012).

8. www.microrao.com/micronotes/antibiotics.pdf (Accessed on July, 2012).

Received on 26.12.2013 Modified on 25.01.2014

Res. J. Pharm. Dosage Form. and Tech. 6(2):April- June 2014; Page 81-84

Sample No.	Media	Colony Characteristics	Code No.
C	Mac Conkey Agar	Dark pink, flat, small, circular	I –C1, I-C2, I-C3, I-C4
	Mac Conkey Agar	Light pink with centred, gummy, dome shaped, circular	II –C1,II-C2,II-C3, II-C4
	Brilliant Green Agar	Cream, small, circular raised	III –C1, III-C2, III-C3, III-C4
	Brilliant Green Agar	Yellow, gummy, large Dome shaped, circular	IV–C1, IV-C2, IV-C3, I V-C4
	Baired Parker’ s Agar	Dark black, small, circular	V –C1, V-C2, V-C3, V-C4
B	Mac Conkey Agar	Dark pink, flat ,small ,circular	I –D1, I-D2, I-D3, I-D4
	Mac Conkey Agar	Light pink with centred, gummy, dome shaped, circular	II –D1, II-D2, II-D3, II-D4
	Brilliant Green Agar	Cream, small, circular raised	III –D1, III-D2, III-D3, IIID4
	Brilliant Green Agar	Yellow, gummy, large Dome shaped, circular	IV –D1, IV-D2, IV-D3, IV-D4
	Baired Parker’s Agar	Dark black, small, circular	V–D1, V-D2, V-D3, V-D4