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3. Results

3.1 Prevalence and confirmation of S. aureus

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During the study, a total of 45 isolated samples from various food contact surfaces were collected. Of these isolates, 12 (26.7%) were obtained from chopping board, 12 (26.7%) from tray, 9 (20.0%) from wok, 6 (13.3%) from plate, and 6 (13.3%) from other surface samples. Among all samples examined, 14 (31.1%) yielded S. aureus are presented in Figure 2. The prevalence of S. aureus from chopping board and tray’s surface was higher than others food contact surfaces. The distribution of isolated S. aureus according to the type of sample is given in Table 2. These 14 samples were positively S. aureus after tested on Mannitol Salt Agar (MSA) test and catalase test. These samples have been further confirmed by gram staining and biochemical test with BPA and coagulase test.

3.2 Identification of S. aureus – biochemical test

S. aureus colonies on Baird Parker agar medium were black and shiny with narrow white margins and surrounded by clear zone extending into the opaque medium (Table 1). These colonies were found to be Gram positive cocci in clusters. The presumptive S. aureus isolates which were isolated from food contact surface areas were confirmed with the biochemical reactions. Whereas Mannitol Salt agar was showing the look for the golden-like sheen colonies developed on the agar (Figure 1). Thereafter, distinct well-separated yellow colonies on MSA were noted and used for further characterization (Shittu et al., 2006). Staphylococci were identified based on Gram reaction (Gram-positive cocci in clumps), catalase and coagulase test (tube method using rabbit plasma).

Figure 1: confirmation of S. aureus on Mannitol Salt agar (MSA) and Baird Parker agar (BPA).

 

Figure 2 shows that of the 31 presumptive S. aureus isolates, 14 (45.2%) were found as positive for catalase and coagulase test. They also positive on reaction toward baird parker agar (BPA) and mannitol salt agar (MSA). According to Holt et al., there isolates approved to be S. aureus.

Figure 2: S. aureus positive based on three food premises from all isolated samples.

 

Table 2

Distribution of S. aureus according to the food contact surfaces area.

Types of the surface

N

%

Chopping board

12

26.7

Tray

12

26.7

Wok

9

20.0

Plate  

6

13.3

Others

6

13.3

Total

45

100

 

 

 

3.3 Antimicrobial resistance test

According to the resistance test results done for all years, all S. aureus isolates were identified as sensitive against Amoxycillin, Penicillin G and Nalidixic acid (Figure 3). Resistance rates against Amoxycillin, Penicillin G and Nalidixic acid were determined as 64.3%, 85.7% and 28.6% respectively. Penicillin G (12/14; 85.7%) and Amoxicillin (9/12; 64.3%) which belong to group Penicillin were determined to be antibiotics with the highest resistant (Table 3). The methicillin resistance rate of S. aureus strains over all the antibiotics was determined to be 27.03% in total.

The isolates were resistant to penicillin (85.7%), amoxicillin (64.3%), and nalidixic acid (28.6%). Furthermore, intermediary resistance to nalidixic acid (26.3%) and susceptible to all antibiotics were observed. Multi-resistance to quinolones and penicillin classes was observed in 16.23% of the isolates. These results are shown in Table 3.

 

 

Figure 3: Diameter of zone inhibition on S. aureus towards antibiotics

 

 

Table 3

Antimicrobial Susceptibility Pattern of S. aureus obtained from food contact surfaces of food premises in Sri Serdang

Isolates

                                           Antibiotics (µg)/Inhibition Zone (mm)

 

CIP 5

AML 10

CN 10

P 10

CRO 5

SF 300

S 25

CAZ 30

KF 30

NA 30

CTX 30

SA001

31.2±0.29

29.3±0.24

21.8±0.85

17.8±0.22

27.5±0.41

19.5±0.45

19.8±0.21

26.2±0.24

30.8±0.71

15.0±0.12

32.2±0.85

SA002

28.5±2.94 

27.8±1.31

26.2±0.85

17.0±1.08

27.7±0.94

17.1±0.29

20.0±0.21

24.5±1.08

31.0±1.08

14.8±0.22

29.8±0.62

SA003

31.7±0.21

37.2±0.62

23.3±0.62

41.7±0.95

28.1±0.76

22.4±0.33

21.8±0.21

25.5±0.41

40.0±0.41

19.9±0.42

32.5±0.41

SA004

31.5±0.37

29.7±0.62

23.8±0.85

18.0±1.08

28.5±0.41

19.9±0.26

19.3±0.21

24.8±0.24

31.3±0.62

15.1±0.26

30.2±0.62

SA005

34.4±0.43

40.5±0.41

26.3±1.02

42.3±2.09

30.5±1.22

30.9±0.29

23.8±0.22

25.8±0.24

43.2±0.85

17.7±1.60

33.8±0.24

SA006

32.1±0.29

26.0±0.41

23.1±0.29

17.8±0.62

27.9±0.42

17.9±0.33

19.2±0.22

25.5±0.41

30.8±1.31

14.8±0.21

26.8±0.24

SA007

32.1±0.29

28.0±0.41

19.8±1.54

18.2±0.62

27.4±1.05

20.0±0.12

18.9±0.14

23.5±0.41

36.7±1.43

15.6±0.29

26.7±0.47

SA008

33.1±0.54

28.5±0.41

20.7±0.85

17.2±0.85

27.2±0.85

21.4±0.49

20.1±0.49

24.8±0.24

31.2±0.21

15.5±0.41

28.5±0.41

SA009

25.8±1.02

33.0±0.41

20.0±1.87

24.7±1.65

21.8±1.56

18.2±0.62

18.6±1.80

18.0±0.82

33.0±1.47

9.1±0.26

24.5±0.71

SA010

31.4±0.66

26.7±0.61

25.5±0.40

22.8±0.21

27.4±1.45

22.3±0.21

24.4±0.49

21.2±1.03

33.4±0.76

17.9±1.80

27.3±0.47

SA011

29.6±0.29

25.7±0.47

17.2±0.85

17.4±0.76

28.8±1.60

20.8±0.21

19.8±0.21

23.0±0.82

32.5±1.83

15.3±0.62

28.2±0.62

SA012

24.8±0.61

26.5±0.41

17.3±0.85

18.2±0.41

26.3±0.92

18.7±0.31

16.8±0.21

23.2±0.62

30.6±0.42

14.5±0.53

26.8±0.24

SA013

30.1±0.29

25.3±0.94

17.7±1.31

18.7±1.03

26.5±0.41

19.6±0.29

20.6±0.42

21.3±1.70

31.0±1.22

15.0±0.12

25.7±0.85

SA014

29.5±0.45

28.5±0.41

21.2±1.55

18.3±0.62

27.3±0.62

18±0.41

19.5±0.52

25.0±0.41

33.6±1.35

15.1±0.29

31.5±0.41

KEY: R=Resistance, Int=Intermediate, S=Susceptible, CIP=Ciprofloxacin, AML=Amoxycillin, CN=Gentamicin, P=Penicillin G, CRO=Ceftriaxone, SF=Sulphafurazole, S=Streptomycin, CAZ=Ceftazidime, KF=Cephalothin, NA=Nalidixic Acid, CTX=Cefotaxime.

 

 

11 antibiotics from five different classes (Penicillin, Sulphonamide, Cephalosporins, Quinolones and Amino-glycosides) have been used against Staphylococcus aureus. The zone of inhibition for each sample towards each antibiotic was measured and data collected as shown as above. Disc diffusion test that showed diameter of inhibition zone present the antibiotic susceptibility toward S. aureus.

Table 2

Antimicrobial resistance of S. aureus isolated from food contact surfaces of restaurants to commonly used antibiotics

Antibiotics

Resistant

Intermediate

Susceptible

 

Positive strain n(%)

Zone diameter (mm)

Positive strain n(%)

Zone diameter (mm)

Positive strain n(%)

Zone diameter (mm)

Ciprofloxacin (5 µg)

00(00)

?12

00(00)

13-14

14(100)

?15

Amoxycillin (10 µg)

9(64.3)

?28

00(00)

   –

5(35.7)

?29

Gentamicin (10 µg)

00(00)

?12

00(00)

13-14

14(100)

?15

Penicillin G (10 µg)

12(85.7)

?28

00(00)

  –

2(14.3)

?29

Ceftriaxone (5 µg)

00(00)

?13

00(00)

14-20

14(100)

?21

Sulphafurazole (300 µg)

00(00)

?12

00(00)

13-16

14(100)

?17

Streptomycin (25 µg)

00(00)

?12

00(00)

13-14

14(100)

?15

Ceftazidime (30 µg)

00(00)

?14

00(00)

15-17

14(100)

?18

Cephalothin (30 µg)

00(00)

?14

00(00)

15-17

14(100)

?18

Nalidixic acid (30 µg)

4(28.6)

?14

10(71.4)

15-22

0(00)

?23

Cefotaxime (30 µg)

00(00)

?14

00(00)

15-22

14(100)

?23

It should be noted that the percentage positive of the strains was from the mean of three replications.

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