Isolation and identification of pathogenic bacteria from Indian milk product-khoa

Introduction

The most common contaminants i.e. E. coli and Staphylococcus pathogenic bacteria could be an important causative agent for gastrointestinal infection including food poisoning and food borne illness. Raw and unpasteurized milk obtained from milch cow and buffaloe by hand milking may contain bacteria from cows udder, flies, manure etc. Indigenous sweet based like khoa is highly susceptible to varieties of microorganisms because of high nutritive value and complex chemical composition (Soomro et al., 2003) [17] . In recent times dairy industry has been concerned by presence of pathogens in dairy products with common sources of contamination as hands, clothing of food processor and dirty surfaces of equipments. A broad spectrum of microbial pathogens can contaminate dairy products and water supplies causing illness after consumption. These includes varieties of enteric bacteria, aerobes and anaerobes (Zagare et al., 2012) [22] . S. aureus is one of the most dangerous and harmful organisms to human health due to its ability to produce enterotoxins associated with food poisoning. The source of contamination is probably human (Kumar and Prasad 2010) [12] . Presence of E. coli in khoa samples is an indicator of faecal contamination (Sohal et al. 1993) [16] . Salmonella enters food chain due to human contamination. Condensed milk product like peda prepared from khoa also gets contaminated by Salmonella (Tambekar and Bhutda 2006) [19] . Spore forming aerobes like Bacillus cereus enters khoa samples through soil contamination. Incidence of Bacillus cereus contamination in milk products is high (Kamat et al., 1989) [9] . Improperly handled dairy products results into contamination of Clostridium perfringens thereby providing means for transmission to consumers (El-Bassiony, 1980) [4] .

Milk and milk products are important sources of Listeria transmission to human being (Rahimi et al. 2010) [15] .

Materials and Methods

Collection of Khoa samples

Khoa samples were collected from khoa market of Parbhani city by following standard method as per FSSAI (2010) [6] . A quantity of 50 gm of Khoa sample was collected in sterile polyethylene sachet. These khoa samples were brought to the laboratory on ice and processed immediately.

Isolation of organisms

Isolation of S. aureus and E. coli, Salmonella spp., Bacillus cereus, Clostridium perfringens, Listeria spp. was done as per the method described in BAM (1998) [1] .

Isolation of Staphylococcus aureus

By using 0.1 ml inoculums of 10 -3 and 10 -4 dilutions of sample on Vogel Johnsen Agar (Hi-Media Laboratories, Mumbai). The inoculums was spread by means of L-shape spreader and plates were kept overnight at 37 o C for incubation. Characteristic colonies of Staphylococci showing typical black colonies were selected. Pure cultures were used for identification by biochemical tests.

Isolation of Escherichia coli

A quantity of 0.1 ml of inoculam from dilution of 10 -3 and 10 - 4 were used by spread plate technique on Levine's Eosine Methylene Blue Agar (EMB) (Himedia Laboratories, Mumbai). Bluish purple coloured colonies with greenish metallic sheen were considered indicative of E. coli. E. coli isolates was maintained on EMB agar for further identification by biochemical test.

Isolation of Salmonella spp.

Pre enrichment was done by using 1 percent Buffered Peptone Water (BPW) (Hi-Media Laboratories, Mumbai) at 37 o C for 24 hours. Pre-enriched sample was inoculated in tetrathionate broth (TTB) (Hi-Media Laboratories, Mumbai) and incubation was done for 24 hours at 42 o C. Streaking of TTB grown bacteria was done on Xylose Lysine Deoxycholate (XLD) (Hi-Media Laboratories, Mumbai) agar and incubated for 24 hours at 37 o C. Pink colonies with or without black centers were considered as positive for salmonella.

Isolation of Bacillus cereus

Khoa sample was inoculated on nutrient broth and then Samples were treated at 70 o C for 15 min. Incubated at 37 o C for 18 hours. Selective plating was done on Bacillus cereus agar (Hi-media Laboratory, Mumbai.) followed by inocubation at 37 o C for 24 hours. Luxuriant growth with blue coloured colonies with precipitation were taken as Bacillus cereus positive.

Isolation of Clostridium perfringens

Inoculated khoa sample in nonselective media (nutrient broth) in 1:10 ratio and homogenized and incubated at 44 o C for 24-48 hours. Taken inoculum from enrichment broth in sterile test tube. To this added sterile molten (50 o C) Sodium Polymixin Sulphadiazine (SPS) as selective agar. (Hi-media Laboratory, Mumbai.) Mixed well and allowed to solidify again added a layer of molten agar over it, allowed to solidify. Incubated in normal incubator at 37 o C for 24-48 hours. A typical colony of Clostridium perfringens on SPS tube showed Black cotton wool like growth.

Isolation of Listeria spp.

Taken at least 10 gm of khoa sample and homogenized with 90 ml of enrichment broth (Fraser-I) (Hi-media Laboratory, Mumbai.) and incubated at 30 o C for 24 hours. Transferred 0.1 ml inoculum from Fraser-I broth to Fraser-II broth(Hi-media Laboratory, Mumbai.) and incubate for 24 hours at 30 o C. From enrichment broth streaked loopful inoculums onto selective Listeria Identification agar (PALCAM) (Hi-media Laboratory, Mumbai.) and incubated plate at 30 o C for 48 hours. On Listeria Identification agar (PALCAM), colonies of Listeria appeared as a grey-green with black center and a black precipitate.

Identification and characterization of Isolates

Identification of S. aureus, E. coli and Salmonella isolates was done by morphological studies by Grams staning, biochemical and sugar fermentation tests.

Morphological characterization of isolates

The S. aureus, E. coli and salmonella isolates were further characterized for morphological characterization by using Gram's staining method. The isolates were inoculated into nutrient broth tubes and incubated for 12 hours at 37˚C. A loopful inoculam from nutrient broth (NB) was streaked on nutrient agar for morphological characters. Gram's staining reaction of each isolate was recorded.

Biochemical characterization of isolates

All S. aureus, E. coli and salmonella isolates were characterized by biochemical tests by using method of Cown and Steel (1993).

Methyl Red test

Pure colony of isolates was inoculated into 0.5 ml sterile glucose phosphate broth and incubated at 37 o C for 12 hours. A drop of methyl red solution was added in each tube and the color change was observed. Developments of red color indicated positive test were as yellow colour was negative test.

Voges -Proskauer (V-P) test

Organism under test was inoculated into 2 ml of sterile Buffered Glucose broth and incubated at 37ºC for 48 hrs. Then 0.2 ml of 40 percent potassium hydroxide was added, followed by 0.6 ml of -naphthol solution. The tubes were shaken vigorously. The cotton plugs were removed and allowed to stand for 5 to 10 min. at room temperature to observe the color formation. Development of pink red colour indicates positive result.

Indole test

The isolates were inoculated in to 3 ml of indole medium (Himedia Laboratories, Mumbai) and incubated at 37ºC for 48 hrs. Then 0.5 ml of Kovacs reagent was added to form a ring in between medium and reagent. Development of bright red colour indicates positive test.

Citrate test

Simmons citrate agar (Himedia Laboratories, Mumbai) slant were streaked with straight wire loop using broth culture and then stab the butt and incubated at 37 o C for 48 hrs. Discoloration of agar to bright blue was considered as positive test.

Nitrate reduction test

Heavy growth of organism was inoculated in 0.5 ml of sterile nitrate broth. Incubation was done at 37 o C for 24 hrs. Then one drop of sulphanalic acid and the -naphthylamine reagent were and observed for colour change. Development of red colour indicates positive reaction.

Sugar fermentation test

Sugar fermentation tests of all isolates were done as per method described by Cruickshank (1975). A 1 per cent sugar solution was prepared and added in Buffered Pepton Water (BPW) with phenol red indicator (pH 8.8). Test organism was inoculated to this medium and Incubated overnight at 37 o C. Presence of yellow colour upon incubation was taken as positive for sugar fermentation. Production of gas was recorded by using Durham's tubes. Appropriate controls were added.

Identification of Bacillus cereus

Bacillus cereus isolates were confirmed on basis of Bacillus cereus agar as a selective agar medium and Nitrate broth reduction.

Results and Discussion

Khoa market in Parbhani city

In Parbhani city, Khoa market is in open ground. Approximately 15 vendors operate in morning hours. The khoa come in market from neighbouring villages in unorganised manner. Each vendor handles approximately 12 Kg of khoa in the market. The khoa is brought to the market in stainless-steel buckets with polythene bags lining and without polythene lining in open containers. The storage of khoa is done at room temperature. The market is operated throughout the year. The conventional Khoa handling, transportation and storage method makes it vulnerable for microbial hazards. In present study microbial quality of khoa was assessed in addition to isolation and identification of various pathogens of public health significance. A total of 15 khoa vendors were identified from market in Parbhani city and given shops codes. A total of 100 khoa samples were collected and analysed in present study.

Isolation of S. aureus

A total of 100 khoa samples were screened for isolation of S. aureus on VJ agar. Samples showing black coloured colonies were considered as positive for S. aureus. The results are shown in Plate 1. A total of 91 S. aureus isolations were done. The isolates were coded for further use. Varadraj and Nambudripad (1982) [21] studied Staphylococcal incidence in market khoa from Banglore and Mysore cities. A total of 125 S. aureus isolations were done. The highest Staphylococcal isolations (56.6 percent.) were done by Masud et al. (1988) [14] from milk and milk products. Many other workers khoa (Teufel et al. 1992 [20,16,17,12] . They observations in present study are on similar lines. Sohal et al. (1993) [16] investigated survival of E. coli in khoa. Heat processing of milk for khoa production eliminated E. coli but during storage growth appeared. Soomro et al. (2002) [18] reported E. coli contamination of khoa. E. coli contamination of pedha was reported from Amrawati city by Tambekar and Bhutda (2006) [19] . Contamination of khoa by E. coli was also reported by many workers (Londhe and Dharampal, 2008; Farzana et al., 2009; Kumar and Prasad, 2010; Zagare et al. 2012; Iqbal and Hussainy, 2014) [13,5,12,22] . In present study a total of 30 E. coli isolates were obtained on EMB agar from khoa samples. The results are given in Plate 2. The results are similar line as reported earlier.

Isolation of E. coli

Plate 1: Details of colonies of S. aureus isolated on VJ agar.

Plate 2: Details of colonies of E. coli isolated on EMB agar

Isolation of Salmonella spp

All 100 khoa samples were screened on XLD agar for Salmonella isolation. A total of 53 isolates were obtained. The results are depicted in Plate 3. Tambekar and Bhutda (2006) [19] studied microbial quality of pedha in Amrawati city. A total of 16.36 percent isolates were identified as Salmonella typhi. A total of 7 Salmonella typhi isolates were reported from milk products by Zagare et al. (2012) [22] . In present study also a total of 53 Salmonella spp. isolates were obtained. The results are on similar lines as reported earlier. [9] used Mannitol egg yolk polymixin B agar for isolation of Bacillus cereus from milk products. Bacillus cereus was present in 28.

Isolation of Clostridium perfringens

Clostridium perfringens was used as an indicator of soil contamination of khoa samples in present study. All the khoa samples (100 No.) were inoculated on SPS agar and incubated at 37 0 C for 24 hrs. The samples showing presence of black cotton wool like growth were taken as positive. A total of 42 isolations were obtained. The details of results are given in Plate 5.

El-Bassiony (1980) [4] screened 163 samples of milk and milk products for Clostridium perfringens isolation on SPS agar. The isolation rate was 45.5 percent in raw milk, 10 percent in cheese, 50 percent in yoghurt and 20 percent in ice cream. Gurmu et al. (2013) [8] successfully isolated 33 Clostridium perfringens isolates from pasteurized milk samples obtained from Guwahati.

In present study also Clostridium perfringens isolation percentage was 42 percent from khoa samples. This may be due to vegetative growth of residual spores of Clostridium perfringens after heat treatment during processing upon storage of khoa samples at room temperature. The findings are on similar line as reported earlier. indicate potential risk of infection with Listeria in people consuming milk and milk products. The highest prevalence of Listeria was found in raw milk (22.6 percent) followed by cheese (18.9 percent) (Rahimi et al., 2010) [15] . In present study also a total of 9 Listeria spp. isolates were obtained on Listeria Identification agar (PALCAM) from screening of 100 khoa samples. The results are given in Plate 6.

Plate 6: Details of colonies of Listeria spp. isolated on PALCAM agar

Identification of isolates by conventional methods:

Grams staining, biochemical test and sugar fermentation test were used to identify S. aureus, E. coli, Salmonella spp. and Bacillus cereus. However confirmation of Listeria spp., Clostridium perfringens, Yeast and Mould was done only upto isolation by using selective media. A total of 91 S. aureus primary presumptive isolates were used for identification of isolates by staining and biochemical characteristics and 28 isolates were identified. A total of 24 E. coli isolates were identified out of 30 presumptive isolates. A total of 26 Salmonella spp. were identified out of 53 presumptive isolation. Nitrate reduction test was used to identify Bacillus cereus isolates. A total of 50 Bacillus cereus presumptive isolates subjected to the test. A total of 8 Bacillus cereus isolates were identified. The highest percentage of isolation was recorded of E. coli (80 percent) followed by Salmonella spp. (49.05 percent), S. aureus (30.76 percent) and Bacillus cereus (16 percent). Earlier Varadaraj and Nambudripad (1982) [21] used biochemical characters like pigmentation, mannitol fermentation, nitrate reduction for identification of S. aureus from khoa samples. In present study also biochemical characters also successfully used for identification of S. aureus.

Identification of E. coli from milk and milk products by biochemical methods was done by earlier workers (Tambekar and Bhutda 2006; Kumar and Prasad 2010) [19,12] . In present study also identification of E. coli from khoa samples was done successfully. Identification of Salmonella spp. isolates from khoa samples was done in present study by using staining and biochemical characters successfully. Earlier also Salmonella spp. identification from milk and milk product was done with the help of staining and biochemical characters (Bandekar et al. 1998, Godbole et al. 2013) [2,7] .

References

1. US Food and Drug Administration. Bacteriological Analytical Manual. 8th ed. Gaithersburg: AOAC International; 1998.
2. Bandekar J, Kamat A, Thomas P. Microbiological quality of the dairy product pedha and its improvement using gamma radiation. Journal of Food Safety. 1998;18(3):221-230.
3. Bhatnagar P, Khan AA, Jain M, Jain SK. Bacteriological study of khoa sold in Gwalior and Morena city (Madhya Pradesh) in relation to public health. Asian Journal of Experimental Sciences. 2007;21(1):55-62.
4. El-Bassiony TA. Occurrence of Clostridium perfringens in milk and dairy products. Journal of Food Protection. 1980;43(7):536-537.
5. Farzana K, Akhtar S, Jabeen F. Prevalence and antibiotic resistance of bacteria in two ethnic milk based products. Pakistan Journal of Botany. 2009;41(2):935-943.
6. Food Safety and Standards Authority of India. Manual of methods of analysis of foods: Milk and milk products. New Delhi: Ministry of Health and Family Welfare, Government of India; 2010.
7. Godbole S, Dabholkar P, Mallik S. Evaluation of bacteriological quality of khoa samples sold in Nagpur city (Maharashtra)-a case study. Journal of Global Biosciences. 2013;2(2):49-52.
8. Gurmu EB, Hazarika RA, Borah P, Barua AG. Presence of enterotoxigenic Clostridium perfringens in foods of animal origin, Guwahati, India. Journal of Environment and Occupational Science. 2013;2(1):45-50.
9. Kamat AS, Nerkar DP, Nair PM. Bacillus cereus in some Indian foods, incidence and antibiotic, heat and radiation resistance. Journal of Food Safety. 1989;10(1):31-41.
10. Karthikeyan N, Dhanalakshmi B. Hygienic quality of Indian sweet milk products from different sources. Bangladesh Journal of Microbiology. 2010;27(2):62-64.
11. Kumar G, Srinivasan MR. Suitability of selected packaging materials for storage of khoa. In: XX International Dairy Congress; 1978; Paris. p. 996-997.
12. Kumar R, Prasad A. Detection of E. coli and Staphylococcus in milk and milk products in and around Pantnagar. Veterinary World. 2010;3(11):495-496.
13. Londhe GK, Pal D. Studies on the quality evaluation of market samples of brown peda. Indian Journal of Dairy Science. 2008;61(6):347-352.
14. Masud T, Bari A, Shah MA. Enterotoxigenicity of Staphylococcus aureus isolated from milk and milk products. Indian Journal of Nutrition and Dietetics. 1989;26(8):239-242.
15. Rahimi E, Ameri M, Momtaz H. Prevalence and antimicrobial resistance of Listeria species isolated from milk and dairy products in Iran. Food Control. 2010;21(11):1448-1452.
16. Sohal S, Bueschat D, Ledward M. Survival and growth of selected microorganisms in khoa during preparation and storage. Journal of Food Safety. 1993;13(3):195-205.
17. Soomro AH, Arain MA, Khashkeli M, Bhutto B, Memon AQ. Isolation of Staphylococcus aureus from milk products sold at sweet meat shops of Hyderabad. Online Journal of Biological Sciences. 2003;3(1):91-94.
18. Soomro AH, Arain MA, Khaskheli M, Bhutto B. Isolation of Escherichia coli from raw milk and milk products in relation to public health sold under market conditions at Tandojam. Pakistan Journal of Nutrition. 2002;1(3):151-152.
19. Tambekar DH, Bhutda SH. Prevalence of bacterial pathogens in pedha (a milk product) sold in Amravati (India). International Journal of Dairy Science. 2006;1(1):32-35.
20. Teufel P, Bryan FL, Qadar F, Riaz S, Roohi S, Malik ZUR. Risks of salmonellosis and staphylococcal food poisoning from Pakistani milk-based confectioneries. Journal of Food Protection. 1992;55(8):588-594.