Infectious Microbes Found In Cows Milk Biology Essay

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Milk is a healthy and nutritional food, (pH 6.7±0.09). It is an aqueous solution of proteins, fats and carbohydrates with presence of many minerals and vitamins.. Milk contains relatively few bacteria when it is secreted from the udder of a healthy animal. However, during milking operations it gets contaminated from the various areas, dairy utensils, milking machines, the hands of the milkers, from the soil and dust. In this way, bacteria, yeasts and molds get into the milk and forms the normal flora of milk. The number of contaminants added from various sources depends on the care taken to avoid contamination

Infectious microbes found in cow's milk

Bacillus cereus: These bacteria produces toxin that causes diarrhea and vomiting. Bacillus cereus have heat-resistant spores and may even survive pasteurization.

Brucella: A bacterial microbe usually found in unpasteurized dairy products. Since there is regular recurrence of fever associated with the disease Brucella infection, or Brucellosis, has also been called "Undulant Fever".

Campylobacter jejuni: It is the most common bacteria that causes diarrheal disease and the chance of disease increses when raw milk consumed, because the basic pH of milk neutralizes the acidity of the stomach.

Coxiella burnetii:. This microbe is resistant to heat and drying and can be found in cow's milk. Infection causes Q fever, a high fever that may last up to 2 weeks.

E. coli O157:H7: This particular strain causes bloody diarrhea and has been associated with a number of food-borne outbreaks. Usually associated with dairy cattle, microbial contamination of raw milk and soft cheeses can result in disease.

Listeria monocytogenes: A common bacterial pathogen found in soft cheeses and unpasteurized milk. Survives below freezing temperatures and therefore withstand refrigeration. Individuals who have weakened immune systems are particularly in dangerous, including pregnant women, AIDS patients, and the very young and very old.

Mycobacterium avium subspecies paratuberculosis: This strain of Mycobacteria can withstand pasteurization and has been associated with inflammatory bowel syndrome or Crohn's disease. However, its actually infection in humans remains controversial.

Mycobacterium tuberculosis: Mycobacterium bovis is in association with consumption of raw milk. M. bovis causes tuberculosis in cows that passes to humans via unpasteurized cow's milk and causes a disease that is very similar to M. tuberculosis.

Salmonella: Several outbreaks in recent years have occurred due to contamination of raw milk and milk products with Salmonella. Diseases symptoms include diarrhea and high fever.

Staphylococcus aureus: A toxin producing microbe that causes explosive vomiting. The disease is a consequence of actual food poisoning from simply consuming the toxin, rather than from an actual infection.

Yersinia enterocolitica: This microbe is associated with raw milk and ice cream, among other foods. A breakdown in sanitization and sterilization techniques at dairy processing facilities can lead to contamination.

Beneficial microorganisms found in cow's milk.

Raw milk and other milk-based products also contain beneficial bacteria. Lactic acid bacteria such as Lactococcuss, Streptococcus lactis, Lactobacillus casei and L.acidophilus, Pediococcus and Leuconostoc help produce yogurt and other dairy foods which have high nutritional value play an important role in promoting gastrointestinal health in humans..

In Sikkim, milk is usually consumed either after boiling or fermented. Milk production is dominated by smallholder farmers, including landless agricultural workers, who rely primarily on family labor to collect and deliver milk to consumers and markets. So during transport of milk from rural areas to urban areas chances of contamination increases.

According to the Sikkim Cooperative Milk Producers Union Ltd. the total milk production in the state is estimated to 31.00 M.T. (thousand) per annum, The native cattle of Sikkim are Siri and Siri-type but artificial insemination and placing of exotic breeding bulls in Veterinary dispensaries and Veterinary hospitals has influenced the native type into the crossbred. Nearly 50% of the population is crossbred the rest are Siri and non-descript type. The crossbreeds present in state are Cattle Jersey, Jersey crossbred, Brown Swiss crossbred, Mixed-cross, Siri, Holstein.

Thus, microbiological analysis of milk is important to find the degree of contamination with the dictions and enumeration of indicator organisms i.e. the coliform group of bacteria which is defined as the indicator (faecal coliform) of suitability of milk for drinking (Standard Method Committee, 1981). Coliform particularly Escherichia coli are mostly used as an indicator in the microbiological analysis of food because these coliform bacteria are able to grow well in a variety of substrates and are able to utilize a number of carbohydrates and some other organic compounds as food for energy.

Thus this study is done to determine the presence of E.coli in raw milk sample of Sikkim and their susceptibility to various antibiotics.

Literature overview

Milk and dairy products is an important item in our food. But these products are very suitable for microbial growth, thus it becomes necessary to know the chemistry of milk, its spoilage, method of preservation, and different dairy products where microbes play a positive rather than negative role. Fresh milk is neutral or slightly alkaline but on souring becomes acid because of the lactic acid formed by bacterial action on lactose. It has a water content of 88% and 12% of solids which constitute of 4.8% sugars, 3.5% fats, 3.1% protein and 0.6% salts (Stewart, 1978). With regard to previous reports it has been found that raw milk appears to be one of the favorite medium for Escherichia coli to exist ( Massa et al 1997)

MATERIALS AND METHODS

CULTURE MEDIA

NUTRIENT AGAR

Peptone 5g

Beef extract 3g

Agar 15g

pH 7.0

ENDO AGAR

Peptone 10 g

Lactose 10 g

Dipotassium phosphate 3.5 g

Sodium sulphite 2.5 g

Basic fuchsin 0.4 g

Agar 15 g

PH 7.5

LACTOSE FERMENTATION BROTH 1X AND 2X* (1000 ml)

Beef extract 3.0 g

Peptone 5.0 g

Lactose 5.0 g

pH 6.9

*for 2x broth use twice the concentration of the ingredients.

BRILLIANT GREEN LACTOSE BROTH (BGLB)

Peptone 10.0 g

Lactose 10.0 g

Bile salt 20.0 g

Brilliant green 0.0133g

PH 7.0

NUTRIENT BROTH

Peptone 5.0 g

Beef extract 3.0 g

PH 7.0g

TRYPTONE BROTH

Tryptone 10g

Calcium chloride(reagent) 0.01-0.03m

Sodium chloride 5.0g

METHYL RED - VOGES PROSKAUER BROTH (MR-VP BROTH)

Peptone 7.0g

Dextrose 5.0g

Potassium phosphate 5.0g

pH 6.9

REAGENTS

KOVAC'S REAGENT (for detection of indole)

p- Dimethylaminobenzaldehyde 5.0g

Amyl alcohol 75.0ml

Hydrochloric acid (concentrated) 25.0ml

METHYL RED SOLUTION (for detection of acid)

Methyl red 0.1g

Ethyl alcohol 300.0ml

Distilled water 200.0ml

BARRITT'S REAGENT (for detection of acetylmethylcarbinol)

Solution A:

α- napthol 5.0g

95% ethyl alcohol 100ml

Solution B:

Potassium hydroxide 40.0g

Distilled water 100.0ml

METHODS

Milk Sampling

For this study total fourteen samples of raw milk were directly collected from different places of Sikkim. The volume of the sample was not less than 100ml and these samples were collected aseptically in sterile Durham bottles , sealed , labelled and stored in 4°C.

Microbial analyses:

The microbial tests considered were standard plate count and most probable number test to determine the presence of coliform bacteria in Sikkim milk.

Coliform test

To determine the presence of coliform in milk sample a statistical estimation method called the most probable number (MPN) test was followed. The most probable number (MPN) test is a multi-step assay comprising of the three steps i.e. presumptive test, confirmed test and completed test. Five tubes of double strength lactose broth and five tubes of single strength lactose broth were inoculated with milk samples measuring 10 ml, 1 ml, and 0.1 ml respectively. During incubation, coliform organisms produce gas. Depending upon which tubes from which milk samples display gas, an MPN index table was consulted and a statistical range of the number of coliform bacteria was determined.

Antimicrobial susceptibility test

Disk diffusion method (Bauer-Kirby method)was used to test the sensitivity of E.coli that was isolated from the samples. This test was performed against eight antibiotics and their concentrations were: erythromycin 15mcg, amoxicillin 30mcg, ampicillin 25mcg, chloramphenicol 30mcg, tetracycillin 30mcg, penicillinG 10units, streptomycin 25mcg, nalidixic acid 30mcg (Himedia).

The agar plates were allowed to solidify. 0.1ml sample from each liquid broth were transferred to the plates and with a help of a spreader, culture was uniformly spread over the agar surface. Using a sterile forceps the antibiotic susceptibility disc was placed over the agar surface and each disc was pressed gently to ensure that the disc adhere to the surface of the agar and the plates were incubated at 37â‚’C for 48hrs. Inhibition zone and bacterial sensitivity against the particular antimicrobial agent i.e. either resistant or susceptible or intermediate based on zone size interpretative chart was observed and recorded.

Characterization of bacterial isolates

Gram staining

It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. A smear of 24hrs old liquid culture was made on a grease free slide. It was then air fixed, heat- fixed, flooded by crystal violet stain for1min,and washed for 5 sec with water. The smear was treated with few drop of Gram's Iodine and allowed to act for a minute. The slide was again washed in water and then decolorized in 95% ethanol. After the smear was decolorized, it was washed with water and the smear was finally treated with few drops of counterstain such as safranin.

Biochemical test

Indole production test

10 drops of Kovac's reagent was added to all the culture broth and the culture were agitated gently. The colour of the reagent layer in each culture was examined after 15-20 mins and recorded in the chart. Appearance of distinct pink or red colour in the upper layer shows positive test.

Methyl red test

(Note:Inoculate tubes of MR-VP broth and incubate for 24hrs at 35°C for Methyl red test and Voges-Proskauer test. The culture broth is divided for the above stated two test)

3-5 drops of methyl red solution was added to the culture broth and the change in the colour was observed and recorded.

Voges-Proskauer test

10 drops of Barritt's reagent 'A' were added and the cultures were shaken in every 3-4 minutes. 15 mins after the addition of Barritt's reagent 'B, the colour of the culture was examined and recorded

RESULTS

Coliform test:

MPN- Presumptive Test for the coliform:

Depending upon the MPN (most probable number) index; Table (1) shows the result for the presumptive test. The milk sample code R1, R2, R4, R6 and R7 shows all the fifteen tubes with positive result thus having the highest probability for the presence of coliforms while R3 and R5 milk sample code shows 12 tubes and 14 tubes with positive results respectively. All the tubes with positive result showed gas production in the Durham tubes.

Table (1): presumptive test for coliform in milk sample

Sample code

No. of tubes with positive results

MPN index

per 100ml

Range 95% probability

Five of 10ml each

Five of 1ml each

Five of 0.1ml each

Lower

Upper

R1

5

5

5

≥2400

700

-

R2

5

5

5

≥2400

700

-

R3

5

4

3

280

100

710

R4

5

5

5

≥2400

700

-

R5

4

5

5

-

-

-

R6

5

5

5

≥2400

700

-

R7

5

5

5

≥2400

700

-

2) MPN-Confirmed test for Escherichia coli:

For the confirmed test, from each gassing positive tubes of lactose broth 1ml of the sample was suspended in Brilliant green broth and incubated for 48hrs. Table (2) shows that milk sample code R1 had all BGLB tubes with positive result while R4, R5 and R6 had nine BGLB tubes with positive result. Sample code R7 & R3 showed six BGLB positive tubes while R2 showed only five tubes with positive result. It was observed that there was decrease in number of tubes with positive result. All these tubes with positive result had gas production and colour of brilliant green broth was changed from green to yellow.

Table (2): confirmed test for Escherichia coli in milk sample

Sample code

Five of 1ml each

Five of 0.1ml each

R1

5

5

R2

5

NIL

R3

4

2

R4

4

5

R5

5

4

R6

5

4

R7

4

2

MPN-Completed test for Escherichia coli:

A loopful sample from each positive BGLB tube was taken and streaking was done on Endo agar for isolation. All samples showed dark centered, flat colonies with or without green metallic sheen. Further these colonies were transferred and suspended in lactose broth which showed positive result with gas production. Colony from Endo agar was transferred in nutrient broth and incubated for 24 hrs at 35°C and was later subjected to Gram staining. The result showed that all culture appeared gram negative non-spore forming rods. All the cultures appearing gram negative rod-shape was tested for IMViC test and result showed that indole and Methyl red (MR) test was positive whereas Voges-Proskauer (VP) test was negative. Table (3) shows the result for the completed test.

Table (3): completed test for Escherichia coli in milk sample

Sample Code

Gram Stain

(morphology

/reaction)

Endo-agar Plate

Lactose Broth

Indole Production Test

Methyl Red Test

Voges-Proskauer Test

R1

Rod shaped &gram negative

+

+

+

+

-

R2

Rod shaped &gram negative

+

+

+

+

-

R3

Rod shaped &gram negative

+

+

+

+

-

R4

Rod shaped &gram negative

+

+

+

+

-

R5

Rod shaped &gram negative

+

+

+

+

-

R6

Rod shaped &gram negative

+

+

+

+

-

R7

Rod shaped &gram negative

+

+

+

+

-

Antimicrobial susceptibility test:

From table (4) it can be observed that isolates from sample code R1, R2, , R4 and R5 is resistance to Erythromycin (E15) while isolate from sample code R6 and R7 are having intermediate susceptibility to E15. Only isolate showing intermediate effect is R3B5 while R3A3 is resistant.

For Nalidixic acid (NA30) all the isolates from sample R1, R2, R3, R4, R5, R6 and R7 are susceptible with larger zone of inhibition.

Maximum sample are susceptible to Tetracycline (T30) while only R1B4 shows resistant and R2A4 with small inhibition zone, R5A3 is intermediate.

All the sample shows resistant to Amoxycillin (Am30) as no zone of inhibition was observed except one R2A1 which shows intermediate susceptibility with very small zone of inhibition.

(Note: Bacteria with intermediate susceptibility may be considered moderately susceptible and they may respond to the particular antimicrobial agent with wide safe dosage range.)

From table (5) it can be observed that all the isolates from each sample is resistant to Penicillin G (P10) because no zone of inhibition was observed.

Large zone of inhibition shows that all the sample are highly susceptible to Chloramphenicol (C30). They also may be susceptible to Streptomycin (S25) while resistant or intermediate to Ampicillin (A25) when inhibition zone is compared.

From both the table (4) and table (5) it is observed that isolates are highly susceptible to four antibiotics i.e. Nalidixic acid (NA30), Tetracycline (T30), Chloramphenicol (C30) and Streptomycin (S25) while resistant to Erythromycin (E15), Amoxycillin (Am30), Penicillin G (P10). For Ampicillin (A25) isolates may be resistant or intermediate.

Table (4): Antimicrobial susceptibility of isolates

Sample code

Isolates code

Diameter of zone of inhibition in mm w/r to Antibiotic

E15

NA30

T30

Am30

R1

R1A2/

-

26mm

22mm

-

Sensitivity

R

S

S

R

R1B4

-

20mm

14mm

-

Sensitivity

R

S

R

R

R2

R2A1

-

27mm

19mm

21mm

Sensitivity

R

S

S

I

R2A4

-

22mm

18mm

11mm

Sensitivity

R

S

I

R

R3

R3A3

-

26mm

20mm

-

Sensitivity

R

S

S

R

R3B5

15mm

26mm

20mm

-

Sensitivity

I

S

S

R

R4

R4A2

-

29mm

21mm

-

Sensitivity

R

S

S

R

R4B4

-

27mm

20mm

-

Sensitivity

R

S

S

R

R5

R5A3

-

23mm

16mm

13mm

Sensitivity

R

S

I

R

R5B5

-

20mm

35mm

-

Sensitivity

R

S

S

R

R6

R6A1

15mm

21mm

24mm

-

Sensitivity

I

S

S

R

R6B5

18mm

22mm

24mm

-

Sensitivity

I

S

S

R

R7

R7A1

13mm

27mm

21mm

-

Sensitivity

I

S

S

R

R7B5

13mm

23mm

24mm

-

Sensitivity

I

S

S

R

Table (5): Antimicrobial susceptibility of isolates

Sample code

Isolates code

Diameter of zone of inhibition in mm w/r to Antibiotic

P10

A25

S25

C30

R1

R1A3

-

10mm

26mm

28mm

Sensitivity

R

-

-

S

R1B5

-

10mm

26mm

28mm

Sensitivity

R

-

-

S

R2

R1A2

-

12mm

26mm

28mm

Sensitivity

R

-

-

S

R1A3

-

18mm

28mm

34mm

Sensitivity

R

-

-

S

R3

R3A4

-

-

22mm

29mm

Sensitivity

R

-

-

S

R3B1

-

-

22mm

28mm

Sensitivity

R

-

-

S

R4

R4A5

-

-

28mm

32mm

Sensitivity

R

-

-

S

R4B3

-

-

26mm

34mm

Sensitivity

R

-

-

S

R5

R5A2

-

13mm

17mm

28mm

Sensitivity

R

-

-

S

R5B4

-

-

30mm

22mm

Sensitivity

R

-

-

S

R6

R6A2

-

-

22mm

28mm

Sensitivity

R

-

-

S

R6B3

-

-

26mm

28mm

Sensitivity

R

-

-

S

R7

R7A2

-

10mm

25mm

33mm

Sensitivity

R

-

-

S

R7B3

-

11mm

26mm

31mm

Sensitivity

R

-

-

S

Conclusions

In this study it has been found that

Standard Method Committee for water and waste water analysis. (1981). J. Ame. Public Health Association Washington .DC.

Cappuccino JG,Sherman N (2007). A laboratory manual in microbiology. 7th edition. Pearson Education Inc. and Dorling Kindersley Publishing, Inc.161-165.

Massa, S., Altiri, C., Quaranta, V. and De Pace, R. 1997. Survival of Escherichia coli O157:H7 in yoghurt during preparation and storage at 4C.Letters in Applied Microbiology 24, 347-350.

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