Prevalence of Escherichia Coli from Raw Fresh Milk Produced from Farm in Negeri Sembilan

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Prevalence of Escherichia Coli from Raw Fresh Milk Produced from Farm in Negeri Sembilan

Aimi Nadia Ramli^1*, Farra Amira Mohamed¹, Noorlis Ahmad¹

1 Faculty of Applied Sciences, Universiti Teknologi MARA Cawangan Negeri Sembilan

*Corresponding Author: [email protected]

Accepted: 1 January 2021 | Published: 15 January 2021

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Abstract: Milk is an extremely nutritious source of food as it contains colloidal suspension of protein, fat, carbohydrate, vitamin and mineral. Drinking raw or undercook milk may harbor the most common foodborne pathogenic bacteria if prepared inexpertly. The aim of this study was to detect the presence and susceptibility level of Escherichia coli towards selected antibiotics in cow raw fresh milk produced on dairy farms in Negeri Sembilan. The cow raw fresh milk samples were randomly selected from Pusat Perkhidmatan Industri Tenusu (PPIT) Kuala Pilah, Negeri Sembilan. Escherichia coli were isolated on Eosin Methylene Blue agar (EMBA) and confirmation was performed by using a series of biochemical test. The resuts revealed that only six out of nine isolates were positive for Escherichia coli, which exhibits a high resistance (100%) towards Vancomycin (VA5). In fact, none of the Escherichia coli isolates were resistant towards Gentamicin (CN10) and Streptomycin (S10). In addition, the multiple antibiotics resistance (MAR) indices were arranged from 0.2 to 0.4. The presence of Escherichia coli in the samples understudy had revealed that there was a high probability of contamination occurred due to the inadequate hygiene condition performed by the farmers while milking, storing and transporting the milk.

Keywords: Raw milk, E.coli, milk contamination, biochemical test, antibiotic susceptibility, MAR Index

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1. Introduction

Milk is an excellent high quality food providing major nutritional requirement to man at any age [1] as it is an aqueous colloidal suspension of proteins, fats and carbohydrates that contain numerous vitamin and minerals [2]. However, an unhygienic production of milk and milk products as well as an improper storage, may cause an early spoilage of microorganisms [3].

Even though raw milk can easily being contaminated with microorganisms, there is still a lack of public awareness among people. Raw milk is still used by large number of farm families and workers and by growing segment of the general population who believe that the milk is not only safe for consumption but also imparts beneficial health effects that are destroyed by pasteurization [4]. Contaminated milk and dairy products have been associated with foodborne illness caused by Salmonella, Brucella, Staphylococcus, Listeria, E. coli and coliforms [3]. The sources of enter- hemorrhagic E. coli strains mostly on foodstuffs as unpasteurized milk and dairy products manufactured from raw milk, have been implicated in many outbreaks [4].

Escherichia coli O157 infection results in diarrhea, thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC) [5]. In Malaysia, the food-borne bacteria such Salmonella, Listeria, Staphylococcus, Campylobacter and E. coli were isolated from animal and animal products [18] [19] [20]. This paper aims to isolate and

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identify the presence of E. coli and to determine an antibiotic susceptibility of E. coli in cow raw fresh milk produced from farm in Negeri Sembilan.

2. Research Methodology

Sampling

TA total of three samples of raw fresh milk were randomly collected from Pusat Perkhidmatan Industri Tenusu (PPIT) Kuala Pilah. During collection, all of the milk samples were placed in a sterile, labelled and cooled sealed plastic bags prior to transportation and were analyze immediately on the day of sampling.

Serial Dilution Method

Serial dilutions were carried out up to 10⁻3 with nutrient broth. A 0.1 ml of each the dilution was transferred to Eosin Methylene Blue Agar (EMBA) plates and was spread using a spreader on the agar surface. Then, the plates were incubated for 18-24 hours at 35°C [6] [7].

Total Plate Count Method

The number of microbial colonies on the selective agar was counted by using the Colony Counter. The expected colony on the EMBA, will be the colony with a metallic greenish color to indicate the presence of E. coli [2]. The numbers of bacterial colonies that grow on the agar surface are count converted to CFU/ml [17].

Biochemical Test

The biochemical tests and morhological characteristics of E. coli on different medium were performed to confirm the colony is E. coli mainly by Gram staining, Methyl-red test, Voges- Proskauer test, Indole test and Simmon Citrate agar test [12].

Antibiotic Susceptibility Test

All isolates under study were tested for susceptibility towards various antibiotics using the disc diffusion method according to guideline set by the National Committee for Clinical Laboratory Standard [13]. The antibiotic susceptibility test was determined based on measurement of inhibition zone [15]. Five different antibiotics such as Imepenem (IPM, 10µ), Tetracycline (TE, 30µg), Gentamicin (CN, 10µg), Vancomycin (VA, 5µg) and Streptomycin (S, 10µg) discs were selected to test against all E. coli isolates [14].

Multiple Antibiotic Resistance Index (MARI)

The multiple antibiotic resistance index (MARI) of the isolates is defined as a/b where ‘a’

represents the number of antibiotics to which the isolate was resistant and ‘b’ the number of antibiotics to which isolate was exposed [8].

3. Findings

In this study, a total of three raw fresh milks were examined for the presence of Escherichia coli. The positive colonies growth of E. coli on EMB agar shows a typical morphological of metallic green sheen colonies [4]. According to Centre for Food Safety (2014), the limitation number of Escherichia coli to be present in raw fresh milk must be lower than 100 to 102 CFU/ml which indicate it is safe to drink. As shown in Table 1, the presence of E. coli were varied from 3.1 x 102 to 1.0 x 105 CFU/ml. However, all raw fresh milk sampled were considered harmful to drink as the number of colonies has exceeded the limitation numbers.

This study showed that, the high prevalence rates of E. coli was in Raw Fresh Milk 1 sample

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with the highest count of 1.0 x 105 CFU/ml and the lowest was 3.1 x 102 CFU/ml in Raw Fresh Milk 3. From our observation, the high bacterial counts in this raw fresh milk sample may be due to unsterile cow’s udder during the milking process or cross-contamination of the laboratory equipment. Moreover, the other factors may be during sampling technique for example temperature abuse during shipping and handling can result in significant growth of E.

coli O157:H7 [10].

Table 1: Detection and microbial load of Escherichia coli in raw fresh milk.

Biochemical tests and morphological characteristics of E. coli on different medium of were performed to confirm it is E. coli by using Gram staining, Methyl-red test, Voges-Proskauer test, Indole test and Simmon Citrate agar test (Table 2). The result shows a same pattern in which two out of three isolated colonies was Escherichia coli. The total of six Escherichia coli were detected from each samples in the 10-2 and 10-3 dilution plated. The 10-1 dilution plated shows a combination of the tests were matched towards Citrobacter koseri as the Citrate test was positive which the color changed from green to blue. The presence of Citrobacter koseri may be due to the cross-contamination such as unsterile condition during the packaging or delivering, infected cow udder, faeces, dust in barns, milk containers or other equipment [11].

Table 2: Confirmation of Escherichia coli in raw fresh milk through IMViC test Sample Dilution

plated

Indole test

Methyl- red test

Voges Proskauer test

Citrate test

Potential bacteria

Escherichia coli + + - -

Raw Fresh Milk 1

10^-1 + + - + Citrobacter koseri

10^-2 + + - - Escherichia coli

The susceptibility of the E. coli isolates against five commonly used antimicrobials was tested and the isolates were characterized as susceptible, intermediate and resistance based on size of inhibition zone [13]. As shown in Table 3, all of the E. coli isolates were susceptible to Gentamicin (100%) and Streptomycin (100%) followed by Imipenem (67%) and Tetracycline (50%). On the other hand, all E. coli isolates were highly resistant to Vancomycin (100%) followed by Imipenem (33%) and Tetracycline (33%). The resistancy showed by Vancomycin had indicate that the antibiotics was ineffective towards the bacteria. Vancomycin apparently

Number of colony (CFU/ml)

10^-1 10^-2 10^-3

Raw Fresh Milk

1 67 (6.7 x 10²) 20 (2.0 x 10⁴) 10 (1.0 x 10⁵) 2 42 (4.2 x 10²) 17 (1.7 x 10⁴) 9 (9.0 X 10⁴)

3 31 (3.1 x 10²) 14 (1.4 x 10⁴) 9 (9.0 x 10⁴)

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able to fight against the bacteria as their mechanism to inhibit cell wall synthesis nonetheless become resistant due to the antibiotic concentration tested was 5 µg. Development of antibiotic resistance among bacteria such as E. coli poses an important public health concern.

Effectiveness of treatments and ability to control infectious diseases may be severely hampered [16].

Table 3: Distribution of antimicrobial resistance, intermediate and susceptible of Escherichia coli isolates No.(%) of Escherichia coli to selected antibiotics

Antibiotics Resistance(R) Intermediate(I) Susceptible(S) Aminoglycosides

Gentamicin (CN10) 0(0) 0(0) 6(100)

Streptomycin (S10) 0(0) 0(0) 6(100)

Beta-lactams

Imipenem (IPM10) 2(33) 0(0) 4(67)

Tetracyclines

Tetracycline (TE30) 2(33) 1 (17) 3(50)

Others (Inhibit cell wall function)

Vancomycin (VA5) 6(100) 0(0) 0(0)

Multiple antibiotic resistance (MAR) indexing has been used as an indicator to identify high- risk contamination potentially hazardous to humans [7]. Multiple antibiotic resistance index value higher than 0.2 was considered to have originated from high risk sources of contamination like human, dairy cattle or commercial poultry farms, while MAR index value equal or less than 0.2 was considered to have originated from strains in animals in which antibiotics were never used [8]. In this study, the multiple antibiotic resistance index (MARI) of the E. coli isolates ranged from 0.20 to 0.40 respectively. About 60% of the isolates possess MAR index of 0.40 and the remainder 40% exhibit MAR index of 0.20 (Table 4). This indicate that the samples are likely represents two different sources of contamination which are low risk (MARI < 0.2) and high risk (MAR > 0.2) sources [8]. Higher value of MAR index specified the microbes were originated from human contamination and were spreading its resistance potential to other aquatic microbes in the aquatic environment [15].

Table 4: Antibiotics resistance patterns and multiple antibiotic resistances (MAR) index of Escherichia coli isolates.

Isolates no. ^aResistance patterns ^bMAR index

R1-10⁻² IpmVa 0.40

R1-10⁻³ IpmVa 0.40

R2-10⁻² Va 0.20

R2-10⁻³ TeVa 0.40

R3-10⁻² Va 0.20

R3-10⁻³ TeVa 0.40

4. Conclusion

The presence of E. coli was known to be a major cause of infections in human and animals as it signified a potential hazard to farmers and consumers. Besides, improper handling of the raw

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cow milk with limited awareness and knowledge on the milk contamination on the public health impact of milk-borne pathogens. In this study, the presence and populations of E. coli as an indicator organisms in raw cow milk samples showed that the produced milk is of poor microbiological quality and of public health risk to the consumers. Based on the results of this study and the similar investigations, it could be concluded that the presence of E. coli bacteria may not necessarily indicate a direct fecal contamination of milk, but it is a precise indicator of poor sanitary practices during milking and further handling process. It is also proven in the study area, there is no standard hygienic conditions followed by producers during milk production besides the quality of water used for cleaning purposes (to wash udder, milk equipment and hands), however, is not secured.

The study also indicated that the E. coli isolates were resistant to most of the antimicrobials used at the study area, which may worsen E. coli infections in the future.

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