Determination of Minimum Inhibitory Concentrations of Antibiotics ...

May-July.2012, Vol.2.No.3, 1358-1362.

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Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences

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Determination of Minimum Inhibitory Concentrations of Antibiotics Effective Against Foodborne Pathogens Isolated from Kulfi Bibhuti Bhushan Sahu1*, R. Usha1, Arijit Das2, Sourav Bhattacharya2 and M. Palaniswamy1 1

Department of Microbiology, Karpagam University, Coimbatore- 641021, Tamil Nadu, India 2

Department of Microbiology, Genohelix Biolabs, A Division of Centre for Advanced

Studies in Biosciences, Jain University, Chamarajpet, Bangalore- 560019, Karnataka, India Received: 28 April 2012; Revised: 25 May 2012; Accepted: 31 May 2012

ABSTRACT Kulfi is a very popular frozen dairy dessert from the Indian Subcontinent. It is available in a variety of flavors and relished by people all over the world. Since kulfi is prepared from ingredients such as milk and water, assessment of its bacteriological quality is necessary to prevent the risk of foodborne infections among the consumers. The present study was undertaken to isolate bacterial pathogens from kulfi sold in Bangalore, India, and to determine the minimum inhibitory concentrations (MIC) of antibiotics effective against them. Eighteen kulfi samples, comprising of six different brands, were analyzed. Different bacterial pathogens were isolated and their biochemical characterization confirmed the presence of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus citreus, Micrococcus luteus, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. The occurrence of psychrotolerant β-haemolytic bacterium Chryseobacterium gleum was also detected in a kulfi sample. Antibiotic susceptibility test revealed that the gram positive bacterial isolates were sensitive to ofloxacin and clindamycin, whereas, the gram negative forms were highly susceptible to ciprofloxacin. In order to determine the MIC, antibiotic concentrations ranging from 0.025-250 µg/ml were used. MIC values of ofloxacin and gentamicin against the gram positive bacteria and that of ciprofloxacin against the gram negative bacterial pathogens were found in the range of 1-5 µg/ml. Higher MIC values observed in the case of some bacterial isolates including the psychrotolerant β-haemolytic bacterium Chryseobacterium gleum may be linked to their increased level of resistance to the tested antibiotics.

Keywords: Minimum inhibitory concentration, antibiotics, foodborne pathogens, kulfi

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Bibhuti Bhushan Sahu et al.

INTRODUCTION A variety of dairy products are produced in India which constitute an important part of Indian cuisine. Kulfi is a popular dessert made by freezing sweetened condensed milk. It is denser than ice cream as it is not whipped. Kulfi is available in different flavors such as cream, chocolate, vanilla, cardamom, saffron, almond, pistachio, mango, etc. It is quite popular due to its widely availability and easy affordability. Unlike other dairy products kulfi has longer keeping quality but may get spoilt due to contamination from bacteria, yeasts and molds. Several factors influence the prevalence and activity of microorganisms in food. Presence of a favorable nitrogen source (milk) and fermentable carbon source (sugar) may facilitate the survival of bacterial pathogens even at freezing conditions. The consumption of such contaminated desserts may result in foodborne illnesses such as typhoid, sore throat etc1. Reports on the assessment of bacteriological quality of kulfi are very few. Some studies conducted earlier reported the presence of various bacterial and fungal pathogens in kulfi sold in Chennai2 and Mumbai3. Selection of suitable antibiotics effective against the pathogens is vital to the treatment of various microbial infectious. Due to rapid emergence and spread of multi-drug resistant bacteria, determination of the minimum inhibitory concentration of an antibiotic is very essential for administration of the correct dose of the antibiotic to the patient4. Therefore, the present study was aimed at isolation and identification of potential pathogens from kulfi sold in Bangalore and determination of the minimum inhibitory concentrations of antibiotics effective against them.

EXPERIMENTAL The present study was conducted at Genohelix Biolabs, Chamarajpet, Bangalore, India. The present investigation constitutes a part of M. Phil. research of the first author admitted to Karpagam University, Coimbatore, Tamil Nadu, India. All the media used during the course of the study were procured from Himedia Laboratories Pvt. Limited (A406, Bhaveshwar Plaza, Mumbai- 400086, India). Sampling of kulfi: Kulfi samples were collected from local vendors and retail shops located at different parts of Bangalore, India. The samples were brought to the laboratory in sterile containers and processed within an hour of procurement. Isolation of foodborne bacteria: Isolation of foodborne bacterial pathogens was performed using serial dilution and spread plate technique. One gram of kulfi sample was properly homogenized in an electric blender. One milliliter of the resultant homogenate was added to 9 ml of sterile 0.85% (w/v) saline in a test tube and serially diluted to obtain dilutions upto 10-5. For bacterial isolation 0.1 ml of the appropriate dilution from each tube was aseptically pipetted out and plated onto different selective and differential media (Tryptic Soy agar, Mac Conkey agar, Cetrimide agar, Baird Parker’s agar, Blood Glucose agar, Tributyrin agar, 10% Salt Milk agar, Eosin Methylene Blue agar, Plate Count agar, Lactobacillus de Mann Rogosa Sharpe agar, Skimmed Milk agar, Bile Esculin agar, Thiosulphate Citrate Bile salts Sucrose agar, Mannitol Salt agar, Polymyxin Egg Yolk Mannitol Bacitracin agar, Mannitol Yolk Red agar, Salmonella Shigella agar, Hichrome UTI agar and Mitis Salivarius Sucrose Bacitracin agar). All the bacterial plates were incubated under aerobic and anaerobic conditions (as per requirement) at 37°C for 24 to 48 h. Identification of the bacterial pathogens: The bacterial isolates were pure cultured, Gram stained and identified by conventional biochemical methods as per standard protocol5 and by 16S rDNA sequencing. Antibiotic susceptibility test: The antibiotic susceptibility test against the bacterial isolates was performed by agar disc diffusion method6. Different standard antibiotic discs such as cefotaxime, ceftazidime, ciprofloxacin, clindamycin, gentamicin, ofloxacin, tobramycin and vancomycin (Himedia, Mumbai, India) were tested. Following overnight incubation at 37°C, diameters of the inhibitory zones were measured to the nearest millimeter using a millimeter scale. 1359 J. Chem. Bio. Phy. Sci. Sec. B, 2012, Vol.2, No.3, 1358-1362.

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Determination of minimum inhibitory concentrations (MIC) of antibiotics effective against the bacterial pathogens: Based on the results of antibiotic susceptibility test, the antibiotics which proved most effective against the bacterial pathogens were selected and their MIC values were determined by broth dilution method7. An initial stock solution of each antibiotic (100 mg/ml) was prepared and diluted by transferring a definite volume into 5 ml of sterile Mueller Hinton broth. Different dilutions such as 250 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml, 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.1 µg/ml, 0.05 µg/ml and finally 0.025 µg/ml were prepared. Having obtained the different concentrations of the antibiotic, each concentration was inoculated with 0.1 ml of the respective bacterial cell suspension and the tubes were incubated at 37°C for 24 h. The growth of the inoculum in the broth was indicated by turbidity or cloudiness of the broth and the lowest concentration of the antibiotic which inhibited the growth of the test organism was considered as the MIC, which was expressed in µg/ml. A negative control was set up by taking only Mueller Hinton broth, while a positive control was maintained with Mueller Hinton broth inoculated with the test bacterial cell suspension.

RESULTS AND DISCUSSION Foodborne illness and food poisoning pose serious threat to both consumers and the food industry8. Several factors play significant roles in causing food spoilage and they are considered as hazards for the food industry. Microbial contamination of foods has lead to numerous outbreaks all over the world. Epidemiological studies have helped in better understanding of the cause of the outbreaks and in implementing precautionary measures to control them9. Anything and everything that comes in contact with food may act as a potent source of contamination. Microbial contamination of foods may occur during any stage of food processing. Therefore, from the selection of ingredients till the transport of the packaged product, strict surveillance is required to ensure the product safety and consumer satisfaction10. Isolation and identification of bacterial pathogens: Eighteen samples of kulfi were purchased from local vendors and retail shops in Bangalore. These samples of different flavors belonged to six different brands. Higher count of isolates was obtained from the samples procured from street side local vendors, while a comparatively low count was observed for the samples obtained from established shops. This may be attributed to the microbial contamination from the dusty surroundings where the desserts are being sold. On the other hand, proper storage under sanitized conditions would have led to reduced bacterial counts in the kulfi sampled from the retail shops. Gram staining revealed the presence of gram positive cocci in clusters, gram positive cocci in short chains, pairs and tetrads, gram positive rods in chains with endospores and gram negative rods scattered. Biochemical characterization of the bacterial isolates confirmed the occurrence of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus citreus, Micrococcus luteus, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Molecular characterization by 16S rDNA sequencing revealed the presence of psychrotolerant β-haemolytic bacterium Chryseobacterium gleum. The prevalence of different species of bacteria may be due to the use of inadequately pasteurized milk and/or raw milk for kulfi preparation11. The processing water might also act as a vector for the transmission of potential pathogens. Raw milk harbors a diverse flora of microorganisms which may be introduced into the milk from the milking animal, personnel, milking utensils and/or processing water. This may explain the predominance of staphylococci and other gram negative bacteria in the kulfi samples which might have appeared from the animal hide or normal microflora of the human skin12, 13. Species of Bacillus might have survived the pasteurization temperature or might have appeared from the sugar used during the preparation. The occurrence of psychrotolerant bacterium Chryseobacterium gleum and βhaemolytic pathogens may be clinically significant due to their ability to cause severe upper respiratory tract infections. Antibiotic susceptibility test: Among the various antibiotics used in this study, vancomycin, ofloxacin, gentamicin and clindamycin were effective against the gram positive bacterial pathogens, with the highest inhibition shown by ofloxacin and gentamicin (data not shown). On the other hand, most of the pathogens exhibited resistance to ceftazidime. In the case of gram negative isolates, ciprofloxacin showed highest 1360 J. Chem. Bio. Phy. Sci. Sec. B, 2012, Vol.2, No.3, 1358-1362.

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inhibition against most of the isolates, whereas, tobramycin, gentamicin and cefotaxime exhibited moderate inhibitory action. Ceftazidime proved less effective against most of the isolates. Determination of minimum inhibitory concentration: To study the efficacy of the antibiotics effective against the bacterial pathogens, MICs were evaluated using broth dilution method14. Antibiotic concentrations ranging from 0.025-250 µg/ml were used. Since ofloxacin and gentamicin largely inhibited the gram positive bacterial pathogens, their MIC values were determined. MIC values of these two antibiotics against all the gram positive test bacteria were evaluated as 2.5 µg/ml, as illustrated in Table 1. Interestingly, the MIC values of ciprofloxacin against the gram negative bacterial pathogens were found in the range of 1-5 µg/ml. Higher MIC value observed in the case of Klebsiella pneumoniae (5 µg/ml) may be linked to its increased level of resistance to the tested antibiotic15. This resistant bacterium may infect the humans via the food chain causing them to suffer from foodborne illnesses4. This may be due to the presence of capsule in addition to the lipopolysaccharide layer in the cell wall which may offer some protection to the bacterium against the inhibitory action of the antibiotic. However, at higher concentration, ciprofloxacin was found to completely kill the pathogen. This antibiotic was found to be highly effective against Pseudomonas aeruginosa as it showed a MIC value of 1 µg/ml. In addition to this, the bacterial cell densities at different concentrations of antibiotics were monitored at 600 nm using a spectrophotometer. Results revealed complete absence of cell growth in the tubes with antibiotic concentrations above the MICs. Table 1. Determination of minimum inhibitory concentrations (MIC) of antibiotics effective against foodborne bacterial pathogens isolated from kulfi Bacterial growth at different concentrations of antibiotic 250* 100 50 25 10 5 2.5 1 0.5 0.1 0.05 Staphylococcus aureus - + + + + Staphylococcus epidermidis - + + + + Staphylococcus citreus - + + + + Micrococcus luteus - + + + + Bacillus subtilis - + + + + Bacillus cereus - + + + + Chryseobacterium gleum - + + + + Escherichia coli - + + + + Klebsiella pneumoniae - + + + + + Pseudomonas aeruginosa - - + + + Bacterial pathogens

MIC 0.025 (µg/ml) + 2.5Of + 2.5Cd + 2.5Cd + 2.5Cd + 2.5Of + 2.5Cd + 2.5Cf + 2.5Cf + 5Cf + 1Cf

Keys: *, concentration of antibiotic in µg/ml; Of, ofloxacin; Cd, clindamycin; Cf, ciprofloxacin; -, Absence of turbidity; +, presence of turbidity

CONCLUSION The prevalence of different bacterial pathogens, especially the psychrotolerant ones in frozen desserts like kulfi, emphasizes the pathological significance of the present study. Prompt identification of the pathogens and proper selection of potent antibiotic(s) are therefore necessary for the treatment of microbial diseases. Determination of correct minimum inhibitory concentration of an antibiotic is important to prevent the development of drug resistance among the microorganisms. It is also suggested that regular inspection of the kulfi quality be practised to avoid the risk of foodborne illnesses among the consumers.

ACKNOWLEDGEMENTS We wish to extend our sincere gratitude to the management of Karpagam University for their encouraging support. Our special thanks to Dr. R. Chenraj Jain, Chairman, Jain Group of Institutions, Bangalore; Dr. N. Sundararajan, Vice-Chancellor of Jain University, Bangalore; Prof. K. S. Shantamani, Chief Mentor, JGI and Dr. S. Sundara Rajan, Director of Genohelix Biolabs, A Division of Centre for Advanced Studies in Biosciences, Jain University, for providing us with the laboratory facilities required for this research work. We also wish to thank Mr. Sandeep Suryan and Mrs. K. Prashanthi from the Department of Molecular 1361 J. Chem. Bio. Phy. Sci. Sec. B, 2012, Vol.2, No.3, 1358-1362.

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Biology, Genohelix Biolabs, Bangalore, for their assistance in molecular characterization. We are also thankful to the entire supporting staff of the laboratory whose help has been invaluable for the successful completion of our research work.

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*Correspondence Author: Bibhuti Bhushan Sahu; Department of Microbiology, Karpagam University, Coimbatore- 641021, Tamil Nadu, India

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