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ORIGINAL ARTICLE
Year : 2012  |  Volume : 2  |  Issue : 1  |  Page : 70-73

Morphological and biochemical characteristics and antibiotic resistance pattern of Staphylococcus aureus isolated from grapes


Department of Biotechnology, Faculty of Engineering, Vinayaka Missions Engineering College, Vinayaka Missions University, Ariyanoor, Tamil Nadu, India

Date of Submission03-Sep-2011
Date of Acceptance14-Sep-2011
Date of Web Publication23-Feb-2012

Correspondence Address:
Sridevi Muruhan
Department of Biotechnology, Faculty of Engineering, Vinayaka Missions Engineering College, Vinayaka Missions University, Salem, Tamil Nadu,
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.93135

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   Abstract 

Aim: To determine the morphological and biochemical characteristics and antibiotic resistance pattern of Staphylococcus aureus isolated from grapes collected from different areas of Bangalore, Karnataka, India. Materials and Methods: Morphological studies of S. aureus by gram staining and further identification by various biochemical tests were performed. Resistance against various antibiotics, including methicillin, and its change under ultraviolet (UV) light was evaluated. Results and Conclusion: S. aureus was isolated and confirmed by the pour plate method and biochemical tests. Pure cultures of the organisms were prepared. The organisms were tested for resistance against ampicillin, tetracycline, kanamycin, and methicillin. The study offers evidence of the presence of S aureus in grapes and provides information on the antibiotic resistance of the organisms.

Keywords: Biochemical test, grapes, isolation, Staphylococcus aureus


How to cite this article:
Konuku S, Rajan MM, Muruhan S. Morphological and biochemical characteristics and antibiotic resistance pattern of Staphylococcus aureus isolated from grapes. Int J Nutr Pharmacol Neurol Dis 2012;2:70-3

How to cite this URL:
Konuku S, Rajan MM, Muruhan S. Morphological and biochemical characteristics and antibiotic resistance pattern of Staphylococcus aureus isolated from grapes. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2019 Jun 17];2:70-3. Available from: http://www.ijnpnd.com/text.asp?2012/2/1/70/93135


   Introduction Top


Staphylococcus aureus is considered the third most important cause of food-borne illnesses in the world. [1],[2]

It is estimated that in the US alone food-borne illnesses affect 6 million to 80 million people each year, causing up to 9000 deaths and costing about 5 billion US dollars. [3] S. aureus is an important cause of food poisoning, pneumonia, wound infections, and nosocomial bacteremia. S aureus can be transmitted by different foods, including milk [4],[5] and beef. [6] However, the antibiotic resistance, especially methicillin resistance, of S. aureus isolated from grapes has not been investigated. Hence, the objective of the present study was to investigate the antibiotic resistance of S. aureus strains isolated from grapes samples collected from various supermarkets.


   Materials and Methods Top


Sample collection

Greenish-white and purple colored grapes were collected from five different places in Bangalore city (Karnataka, India), i.e., Jayanagar, Madiwala, Silk Board, Singsandhra, and BTM layout. Damaged grapes, identified on the basis of the physical appearance (broken skin, shriveled, discolored, etc.), were removed.

Isolation and preparation of pure culture

S. aureus was isolated from grapes. [7] From each plate, typical colonies of S. aureus with similar morphologies were isolated and cultured separately on slants.

Morphological and biochemical studies of S. aureus

After culture and bacterial isolation, the isolates were identified by morphology of colony and gram staining and by standard biochemical tests. [8]

Antibiotic susceptibility test

S. aureus -positive samples were selected for susceptibility tests. Antimicrobial susceptibility testing was done by the Kirby-Bauer disc diffusion method using LB agar medium, according to the Clinical Laboratory Standards Institute guidelines. [9] The following antimicrobial-impregnated discs (HiMedia Laboratories) were used: Tetracycline (15 μg), ampicillin (10 μg), kanamycin, gentamycin, and methicillin. After incubation at 42°C for 48 h in a microaerophilic atmosphere, the susceptibility of the S. aureus to each antimicrobial agent was measured.

Changes by ultraviolet method

The samples that showed resistance to antibiotics were treated under ultraviolet (UV). Sterilized LB broth was taken in five Petri plates and allowed to solidify. Then the sample was added. The five plates were kept for varying periods (plate 1 for 5 min, plate 2 for 10 min, plate 3 for 15 min, plate 4 for 20 min, and plate 5 for 25 min) under UV.


   Results and Discussion Top


The results of this study showed the presence of S aureus in grapes from different places of Bangalore. [Figure 1] shows the formation of a S. aureus colony in the pour plate method. Organisms from grapes streaked on mannitol salt agar showed colonies that fermented mannitol and appeared golden yellow. Morphological characteristics showed the organism to be gram positive, spherical S. aureus.
Figure 1: Formation of S. aureus colony in the pour plate method

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After 24 h, the organism was grown from purple grapes collected from four places and green grapes collected from two places (P1 - Jayanagar juice shop, P2 - Silk Board, P3 - Jayanagar market, P4 - Madiwala juice shop, G1 - Jayanagar juice shop, and G2 - BTM layout). The colony formation in plates can be seen in [Figure 1]. These colonies were further used for pure culture.

The isolated organism showed negative results for indole, methyl red, Voges-Proskauer reaction, hydrogen sulphide, gelatin liquefaction, and oxidase test and showed positive results with citrate, lactose, sucrose, glucose, lipid hydrolysis, catalase, and starch hydrolysis test [Table 1]. Similar biochemical results have been reported for S. aureus.[10]
Table 1: The biochemical test results of S. aureus isolated from grapes

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Both purple (P1-4) and green (G1-2) grapes showed the same results.

The source of the samples was as follows: P1 - Jayanagar juice shop, P2 - Silk Board juice shop, P3 - Jayanagar market, P4 - Madiwala juice shop, G1 - Jayanagar juice shop, and G2 - BTM layout.

Bacterial resistance to antimicrobial agents is a growing problem. This is due to the alteration of resistance mechanisms, acquisition of resistance genetic element from other bacteria, and genetic changes in the bacteria. [11] S. aureus isolated from purple grapes showed more resistance to methicillin than to other antibiotics [Figure 2]. [12] Since methicillin resistance emerged, methicillin-resistant S. aureus (MRSA) has become widespread in hospitals worldwide, causing bacteremia, pneumonia, surgical site infections, and other nosocomial infections. [13] The S. aureus strains isolated from purple grapes (P1-4) and green grapes (G1) in our study were also resistant to methicillin. of purple grapes (P1) also showed resistance to kanamycin and ampicillin [Table 2]. Similar results have been reported by others. [14],[15],[16]
Figure 2: Resistance to ampicillin, methicillin, and kanamycin

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Table 2: The susceptibility of S. aureus to the different antibiotics

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After 24 h of incubation, among the six samples only one (P1) showed resistance to three antibiotics (methicillin, ampicillin, and kanamycin). The remaining showed resistance to methicillin but were sensitive to the other antibiotics.

As P1 showed resistance to three antibiotics, it was further subjected to mutational change study by the UV method. There was growth of S. aureus [Figure 3] in the plates after exposure to UV rays for different time intervals (5 min, 10 min, 15 min, 20 min, and 25 min (named as U1, U2, U3, U4, and U5, respectively). In some plates the growth of the organism was at the corners of the plate, while in others growth occurred all around the plate.

[Figure 4] shows antibiotic susceptibility test and [Table 3] shows the zone of inhibition.
Figure 3: (a) S. aureus growth after UV treatment (5 min) (b) 10 min, 15 min, 20 min and 25 min after UV treatment

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Figure 4: Antibiotic susceptibility test

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Table 3: Zone of inhibition of organisms (in cm)

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S. aureus is one of the most frequent bacteria isolated from raw milk, meat and poultry products. But with the extensive research on the application of technologies such as pulsed electric fields (PEF), high hydrostatic pressure, UV irradiation and ultrasound [17],[18],[19] in industrial-scale food processing has shown that these technologies can combine the advantage of preserving 'freshness' with acceptable inactivation of spoilage-causing and pathogenic microorganisms.

Various studies have demonstrated the presence of fungal, yeast, and bacterial species [20],[21] as well as the mycotoxin ochratoxin A [22],[23] in grapes. Our study shows the multiple antibiotic-resistance exhibited by a strain of S. aureus isolated from grapes.


   Conclusion Top


Further research is needed for molecular characterization of the S. aureus strains.


   Acknowledgment Top


The authors thank Dr. M. Mahesh, MSc, M.Phil, PhD; CEO, Azyme Biosciences, Bangalore, India, for his support in the work.

 
   References Top

1.Asperger H, Zangerl P. Staphylococcus aureus. In: Roginski, H, Fuquay JW, Fox PF, editors. Encyclopedia of Dairy Sciences. Vol. 4. Amsterdam, Boston, London, New York, Oxford, Paris, San Diego, San Francisco, Singapore, Sydney, Tokyo: Academic Press and Elsevier Science; 2003. p. 2563-9.  Back to cited text no. 1
    
2.Boerema JA, Clemens R, Brightwell G. Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus. Int J Food Microbiol 2006;107:192-201.  Back to cited text no. 2
    
3.Balaban N, Rasooly A. Staphylococcal enterotoxins. Int J Food Microbiol 2000;61:1-10.  Back to cited text no. 3
    
4.Citak S, Varlik O, Gundogan N. Slime production and DNAse activity of Staphylococci isolated from raw milk. J Food Saf 2003;5:281-8.  Back to cited text no. 4
    
5.Scherrer D, Corti S, Muehlherr JE, Zweifel C, Stephan R. Phenotypic and genotypic characteristics of Staphlococcus aureus isolates from raw bulk-tank milk samples of goats and sheep. Vet Microbiol 2004;101:101-7.  Back to cited text no. 5
    
6.Schlegelova J, Napravnikova E, Dendis M, Horvath R, Benedik J, Babak V, et al. Beef carcass contamination in slaughterhouse and prevalence of resistance to antimicrobial drugs in isolates of selected microbial species. Meat Sci 2004;66:557-65.  Back to cited text no. 6
    
7.Food and Drug Administration. Bacteriological analytical manual. 7 th ed. Gaithersburg, MD: AOAC International; 1992.  Back to cited text no. 7
    
8.Bergey, David H, Holt JG, Krieg NR, Peter HA. Sneath. Bergey's Manual of Determinative Bacteriology. 9th ed. Philadelphia: Lippincott Williams and Wilkins; 1994.  Back to cited text no. 8
    
9.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests. Approved standard. 9 th ed. (M2-A9). Wayne, PA: Clinical and Laboratory Standards Institute; 2006.  Back to cited text no. 9
    
10.Adejuwon AO, Ajisebutu SO. Effect of sodium chloride on microbial lipase activity on vegetable oil. Afr J Med Pharm Sci 2004;8:11-6.  Back to cited text no. 10
    
11.Hrabak J, Zemanova A, Chudackova E. Mobile genetic elements in the epidemiology of bacterial resistance to antibiotics. Epidemiol Microbiol Imunol 2010;59:55-66.  Back to cited text no. 11
    
12.Deepa S, Kumari AB, Venkatesha D. Increasing Trends of Methicillin Resistant Coagulase Negative Staphylococcus in Neonatal Septicaemia - A Study in a Tertiary Care Hospital, Mysore, South India. Online J Health Allied Sci 2010;9:11.  Back to cited text no. 12
    
13.Wenzel RP, Bearman G, Edmond MB. Screening for MRSA: A flawed hospital infection control intervention. Infect Control Hosp Epidemiol 2008;29:1012-8.  Back to cited text no. 13
    
14.Ihsan Edan Alsaimary. Efficacy of some antibacterial agents on Staphylococcus aureus isolated from various burn Cases. Int J Med Med Sci 2009;1:110-4.  Back to cited text no. 14
    
15.Kurjogi MM, Sanakal RD, Kaliwal BB. Antibiotic susceptibility and antioxidant activity of Staphylococcus aureus pigment staphyloxanthin on carbon tetrachloride (ccl4) induced stress in swiss albino mice. Int J Biotechnol Appl 2010;2:33-40.  Back to cited text no. 15
    
16.Arias, Cesar A, Murray BE. Antibiotic-Resistant Bugs in the 21 st Century - A Clinical Super-Challenge". N England J Med 2009;360:439-43.  Back to cited text no. 16
    
17.Dunne CP, Kluter RA. Emerging non-thermal processing technologies: Criteria for success. Aust J Dairy Technol 2001;56:109-12.  Back to cited text no. 17
    
18.Raso J, Barbosa-Canovas GV. Non thermal preservation of foods using combined processing techniques. Crit Rev Food Sci Nutr 2003;43:265-85.  Back to cited text no. 18
    
19.Ross AI, Grifths MW, Mittal GS, Deeth HC. Combining nonthermal technologies to control foodborne micro organisms. Int J Food Microbiol 2003;89:125-38.  Back to cited text no. 19
    
20.Fleet GH. Yeast interactions and wine flavour. Int J Food Microbiol 2003;86:11-22.  Back to cited text no. 20
    
21.Fleet GH, Prakitchaiwattana C, Beh AL, Heard GM. The yeast ecology of wine grapes. In: Biodiversity and Biotechnology of Wine Yeasts, Ciani M, editor. Kerala, India: Research Signpost; 2002. p. 1-18.  Back to cited text no. 21
    
22.Cabanes EJ, Accensi F, Bragular MR, Abrca ML, Castella G, Minguez S, et al. What is the source of ochratoxin A in wine? Int J Food Microbiol 2002;79:213-5.  Back to cited text no. 22
    
23.Sage L, Krivobok S, Delbos E, Seigle-Murandi F, Creppy EE. Fungal flora and ochratoxin A production in grapes and musts from France. J Agric Food Chem 2002;50:1306-11.  Back to cited text no. 23
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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