 *Corresponding author.

Email: norainy@upm.edu.my

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (2) : 1234 - 1244 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Detection of enterotoxin gene (sea) and biofilm formation ability among multi-

drug resistant Staphylococcus aureus isolated from shawarma sandwich sold at

selected kiosks in Klang Valley, Malaysia

1Salahaldin Fathalla, M. 1,2,*Mahyudin, N.A., 3Mohamad Ghazali, F., 3Rukayadi, Y. and
4Jaafar, S.N.

1Department of Food Service and Management, Faculty of Food Science and Technology,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

3Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400

UPM Serdang, Selangor, Malaysia
4School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu,

Terengganu, Malaysia

Article history:

Received: 30 January 2020

Received in revised form: 14

March 2020

Accepted: 22 March 2020

Available Online: 16 April

2020

Keywords:

Biofilm formation,

Multidrug resistance,

Staphylococcus aureus,

sea genes,

Shawarma

DOI:
https://doi.org/10.26656/fr.2017.4(4).039

Abstract

The occurrence of multi-drug resistant Staphylococcus aureus in food product of animal

origin has increased the concern about their spread into the food supply chain. Presence of

multidrug-resistant S. aureus in food products, including ready-to-eat foods imposes

potential hazard for consumers. The objective of this research was to investigate the

presence of multi-drug resistance of S. aureus in sixty ready-to-eat shawarma sandwiches.

Agar-disc diffusion assay determined their resistance to 11 antibiotics. The sea and sed

enterotoxin genes were detected by polymerase chain reaction method. Biofilm formation

potential (BFP) was quantified by microtitre plate assay. The result revealed that thirty-six

samples (60%) were positive for S. aureus. Majority of the isolates (n = 29; 80.6%) were

resistant to at least one antibiotic. The isolates demonstrated highest resistance against

ampicillin (69.4%) and penicillin (69.4%), while resistance to ciprofloxacin, tetracycline

and kanamycin were 47.2%, 33.3% and 22.2%, respectively. Several isolates were

resistant to trimethoprim (5.6%), trimethoprim-sulfamethoxazole- (2.8%), gentamicin

(2.8%) and cephalothin (2.8%), while none exhibited resistance to chloramphenicol and

nitrofurantoin. Out of the thirty-six isolates, twelve isolates (33.3%) were resistant to three

or more classes of antibiotic (multidrug-resistant) and 50% had a Multiple Antibiotic

Resistance index value more than 0.25. Of the multi-drug resistant isolates, four were

positive for sea genes but no sed genes were present. All multi-drug resistance isolates

were biofilm formers with five and six isolates were strong and moderate formers,

respectively. Additionally, all the sea gene carrying multi-drug resistance isolates were

strong biofilm formers. These findings revealed shawarma as a potential vehicle for the

spread of multidrug-resistant S. aureus, suggesting more control measures for ready-to-eat

food.

1. Introduction

Shawarma sandwiches are served in several fast-

food restaurants in Europe, the Middle East, Canada, and

most recently in Malaysia. Shawarma’s origin can be

traced back to Turkey, and to the Turkish word

“çevirme” meaning to rotate (Sirkeci, 2016). It is

however known in various countries by various names

including chawarma, doner kebab, donair, and gyro

(Ayaz et al., 1985; Todd et al., 1986; Nimri et al., 2014).

Shawarma is a famous sandwich made of chicken, lamb,

or beef. The majority of restaurants serving shawarma

use the breasts of chicken, as these cuts are cheaper and

more easily digestible than red meat (Kayaardi et al,

2006).

Shawarma is mostly popular as street food, so it is at

risk of contamination by food pathogens from the

environment. Among the major problems facing

developing nations including Malaysia are foodborne

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infections caused by microbial agents, likely occurring as

a result of a change in decision-making involving the

production, consumption, and storage of food brought

about by the liberation and globalization of trade and the

increased importance of food (Satcher, 2000; Abdelgadir

et al., 2009; Shafizi et al., 2016). In previous study, an

increasing incidence of foodborne infections had been

observed in Malaysia between 2005 and 2008 (Rahman,

2019). Nevertheless, death rates due to these foodborne

infections have reportedly remained the same with an

equivalent rate of mortality to that obtained in 2008

(Abdul-Mutalib et al., 2012). Additionally, in 2006, 3625

food premises out of a total available of 81,686 were

shut down because they did not meet the requirements of

the Food Act 1983, Section II, as enforced by the

relevant authority (MOH, 2006; Sani et al., 2014). The

foodborne infections in Malaysia can mainly be traced to

unhygienic food handling procedures, which contribute

to 50% of the cases (MOH, 2007).

Shawarma is a food that can potentially become

contaminated with pathogenic bacteria during

preparation and processing. Studies analyzing the

pathogen contamination of shawarma in various

countries revealed that the shawarma had high

contamination of several microbes and certain foodborne

pathogens that were multidrug-resistant including S.

aureus (Harakeh et al., 2005; Odu and Akano, 2012;

Abdalhamid et al., 2013; Nimri et al., 2014; Salem et al.,

2016).

S. aureus is one of the most vital species of bacteria

in food microbiology and has long been regarded as a

potential threat to foodborne illnesses. It colonizes

human skin and the front nares in a substantial fraction

of the human population (Kluytmans et al., 2005; Castro

et al., 2016). Some of these pathogens are resistant to

antimicrobials including the commonly used classes of

antibiotics (Sharaf et al., 2012; Nimri et al., 2014;

Bantawa et al., 2019).

The main threat of food contamination to general

public health stems from bacteria that are resistant to

antibiotics since the determinants of the resistance could

be transferred to other bacterial pathogens in food.

Several reports have shown the occurrence of foodborne

pathogenic microbes that are resistant to antimicrobials

(Sharaf et al., 2012; Nimri et al., 2014).

S. aureus is able to produce heat-stable enterotoxins

responsible for several diseases (Bergdoll, 1983). There

are five serological staphylococcal classical antigens

identified including staphylococcal enterotoxins (SE) A,

B, C, D and E out of which SEA and SED a responsible

for about 95% of the global incidence of staphylococcal

food poisoning (Sezer et al., 2015). The most common

toxin detected in outbreaks of food poisoning in the

United States was the SEA with almost 77% occurrence,

followed by SED with 38% and SEB with 10%. SEA is

the most commonly found enterotoxin among SFD

outbreaks in Japan, France, and UK (Balaban and

Rasooly, 2000; Argudín et., 2010).

To the best of the author’s knowledge, the current

study is the first to determine the occurrence of

multidrug-resistant S. aureus from chicken shawarma in

Arabic kiosks in Malaysia. It further aims to investigate

the prevalence of antimicrobial resistance in the isolates

against some commonly used antibiotics. The findings of

this research will provide the necessary information for

relevant authorities to manage public health threats

associated with the consumption of shawarma

sandwiches.

2. Materials and methods

2.1 Sample collection

A total of sixty shawarma samples were purchased

from kiosks at Arabic restaurants (n=20) in the Klang

Valley area, Malaysia. The samples were obtained from

three visits to each kiosk in the periods of July 2016 to

Mac 2017. Samples were collected in a sterile plastic bag

and kept in an icebox (4-8°C) immediately after

collection and transported to the laboratory within 4

hours.

2.2 Sample processing

Shawarma sandwich samples were cut into smaller

pieces using a sterile knife and 25 g of the samples were

mixed with 225 mL of sterile buffered peptone water

(Oxoid, United Kingdom) in a stomacher bag and

homogenized for 60 s using a Stomacher Lab-Blender

400 (Seward Medical, UK). The homogenized samples

were incubated overnight at 37ºC.

2.3 Isolation and identification of Staphylococcus aureus

An aliquot of 0.1 mL of the homogenized sample

was spread onto a Baird-Parker agar (BPA) medium

(Himedia, India) supplemented with Egg Yolk Tellurite

Emulsion (Himedia, India). After incubated at 37°C for

24 hrs, a typical colony (black/dark gray colony

surrounded by an opaque halo) was selected and

subjected to Gram Staining, catalase test and coagulase

reactions (Lancette and Tatini, 1992). The suspected S.

aureus colonies were further tested by PCR for

confirmation using specific primer pairs of nuc gene

(Integrated DNA Technologies, Inc., Coralville, IA,

USA) with the forward sequence of 5'-

GCGATTGATGGTGATACGGTT-3' and reverse

sequence of 5'-AGCCAAGCCTT GACGAACTAAAGC

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-3' (Brakstad et al., 1992; Kuźma et al., 2003; Yang et

al., 2007). These primers amplify 270 bp region of nuc

gene fragment of S. aureus.  A total of 25 µL reaction

mixture was prepared for the amplification and it

contained 1 µL of 50 ng of the genomic DNA, 1 µL each

of 10 µM of the forward and reverse primers, 12.5 µl of

2x PCRBIO Taq Mix Red (PCR Biosystems Ltd,

London, UK) and finally made up to 25 µL with sterile

distilled water. The amplification was performed in a

thermal cycler (Kyratec Super Cycler Thermal Cycler,

Australia) with the following conditions: initial

denaturation at 95°C for 10 mins followed by 37 cycles

of denaturation at 94°C for 60 s, annealing at 55°C for

30 s, extension at 72°C for 90 s and a final extension 72°

C for 5 mins. S. aureus ATCC 13565 was used as a

positive control in this experiment. For PCR products

visualization, 5 µL of the PCR products were loaded

onto an ethidium bromide-stained 1.5% agarose gel for

electrophoresis and visualization of the amplified PCR

products. The amplicons were then measured at 270 bp

using Gelpilot 100 bp DNA ladder (Qiagen, Hilden,

Germany).

2.4 Antimicrobial susceptibility testing

Susceptibility of all the S. aureus isolates to

antimicrobials was determined by an agar diffusion test

using commercially available antibiotic disks (Oxoid,

United Kingdom) as described by the Clinical

Laboratory Standards Institute (CLSI) guidelines (CLSI,

2017). An overnight Mueller Hinton broth (MHB)

(Merck, Germany) culture of the test isolate (0.5

McFarland) was spread on a Mueller Hinton Agar

(MHA) (Merck, Germany) plate. Antibiotic discs were

evenly dispensed on the inoculated MHA plates and

incubated at 37°C for 18–24 hrs. Eleven types of

antibiotics used were Penicillin (10 µg), Ampicillin (10

µg), Gentamicin (10 µg), Kanamycin (30 µg),

Tetracycline (30 µg), Chloramphenicol (30 µg),

Trimethoprim (5 µg), Sulfamethoxazole-Trimethoprim

(25 µg), Ciprofloxacin (5 µg), Cephalothin (30 µg), and

Nitrofurantoin (300 µg). After incubation, the diameter

of the inhibition zone was measured. Antibiotic

susceptibility profile was determined based on the CLSI

standard (CLSI, 2017). The resistance of a single isolate

to three or more classes of antibiotics (Oteo et al., 2005;

Geidam et al., 2012) was characterized as multidrug

resistance (MDR). The values of multiple antibiotic

resistance (MAR) index were calculated as the ratio of

number of antibiotics to which the organisms were

resistant to the total number of antibiotics to which the

organisms were exposed and is mathematically

represented thus;

MAR = A/B

Where A = the total number of antibiotics to which the

organism is resistant, and B = the total number of

antibiotics to which the organism was exposed

The MAR index is an important tool for the

assessing the health risks that aids in identifying an

isolate originating from a region excessive usage of the

antibiotic (Davis and Brown, 2016). Values of MAR

index greater than 0.2 are considered an important

indicator for assessing the contamination risk

(Krumperman, 1983).

2.5 Detection of enterotoxin genes (sea and sed)

Primers employed for SEA detection consisted the

forward 5’-CCT TTG GAA ACG GTT AAA ACG-3’

and the reverse 5’-TCT GAA CCT TCC CAT CAA

AAA C-3’ with estimated amplicon size of 127 bp, while

primers used for SED detection consisted of the forward

5’-CTA GTT TGG TAA TAT CTC CTT TAA ACG-3’

and the reverse 5’-TTA ATG CTA TAT CTT ATA

GGG TAA ACA TC-3’ with estimated amplicon size of

319 bp (Becker et al.,1998). PCR products were

prepared in a 25 µL reaction volume that consisted of 1

µL of 50ng genomic DNA, 1 µL each of 10 µM forward

and reverse primers, 12.5 µL of 2x PCRBIO Taq Mix

Red (PCR Biosystems Ltd, London, UK) which

composed of PCRBIO Taq DNA polymerase, 6 mM

MgCl2 and 2 mM dNTPs and 9.5 µL sterile distilled

water. Amplification of SEA and SED PCR products

was carried out in a thermal cycler (Model SC300,

Kyratec Australia). The thermal cycler conditions of

amplification include; 94ºC for 5 mins of initial

denaturation, 30 cycles of 94ºC for 1 min of final

denaturation, annealing at 55ºC for 1 min and extension

at 72ºC for 1 min. Finally, extension followed at 72ºC

for 5 mins.

2.6 Biofilm formation

The biofilm formation ability of all the multi-drug

resistant isolates was estimated using the crystal violet

staining method described by Beehan (Beehan et al.,

2015) with minor modifications. An overnight culture of

individual isolate grown in tryptic soy broth (TSB)

(Oxoid) at 37oC was adjusted to 0.5 McFarland and were

diluted in TSB supplemented with 0.25% glucose (1:40).

An aliquot of 200 mL of the dilutions was dispensed in

each well of a sterile polystyrene 96-well microtitre plate

(SPL, Life Science, Korea) and the plates were incubated

at 37ºC for 24 hrs. Following incubation, the wells were

rinsed three times using phosphate buffered saline (PBS;

pH 7.4±0.1; Thermo Fisher Scientific, USA), air-dried

for 1 hr at 60ºC and stained with 0.25% crystal violet

(Thermo Fisher Scientific, USA). The plates were then

incubated at 25ºC for 15 mins and the biofilm formation

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capacity was characterized by measuring the optical

density at OD570nm with a microplate spectrophotometer

(Benchmark Plus Microplate Spectrophotometer System,

Bio-Rad, USA).

Interpretation of the results of the mean absorbance

of the test wells was done in accordance with Stepanović

et al. (2000) as non-adherent, weak, moderate or strong

biofilm formers on the basis of comparison with the

control wells (uninoculated wells) of the plate. Non-

adherent were those with absorbance less than or equal

to the control wells (OD ≤ ODC); the weakly adherent

(ODC ≤ OD ≤ 2x ODC), moderately adherent (2x ODC ≤

OD ≤ 4x ODC) and the strongly adherent (4x ODC <

OD). Isolates were analyzed for the biofilm in triplicates.

Growth medium without bacterial inoculum served as a

negative control and S. aureus ATCC 6538 was used as a

reference strain.

3. Results

3.1 Detection of Staphylococcus aureus in shawarma

sandwich

Among the total 156 isolates obtained from sixty

shawarma samples, thiry-six (23.1%) were positive for S.

aureus (Table 1). More than 25.0% of the positive S.

aureus isolates were detected from shawarma sold in

nine (9) kiosks namely; R-16 (50.0%), R-7 (40.0%), R-6

and R-13 (33.3%), R-12 (30.0%), R-2 (28.5%), R-1, R-

10 and R-19 (25%) while the remaining 11 kiosks had

lower number of positive isolates (<25%).

3.2 Susceptibility of Staphylococcus aureus to antibiotics

Table 2 shows that all 36 isolates (100%) were

susceptible to chloramphenicol and nitrofurantoin. The

isolates demonstrated highest resistance (69.4%) against

both antibiotics in the Penicillin class; namely ampicillin

and penicillin, while resistance to ciprofloxacin,

tetracycline and kanamycin were 47.2%, 33.3% and

22.2%, respectively. Only several isolates were resistant

to trimethoprim (5.6%), trimethoprim-sulfamethoxazole

(2.8%), gentamicin (2.8%) and cephalothin (2.8%).

Intermediate resistance was only observed in 6 isolates

(16.7%) against kanamycin. Of the 36 isolates, 29 were

resistant to at least one (1) antibiotic (see Table 3).

Twelve of the 36 isolates (33.3%) were resistant to three

or more classes of antibiotic (MDR) and 50% had a

MAR index value more than 0.25.

3.3 Characteristic of MDR Isolates of Staphylococcus

aureus, the presence of sea and sed genes and their

biofilm formation potential

 Multidrug resistance associated with enterotoxigenic

genes detection and biofilm formation ability is shown in

Table 4. The main MDR profiles of the isolates were

penicillin- fluoroquinolone-aminoglycosides-tetracycline

(n=6), penicillin-fluoroquinolone-tetracycline (n = 2) and

penicillin-aminoglycosides-tetracycline (n = 2). Four of

the MDR were positive for sea genes but no sed genes

were present in all MDR isolates (Figures 1 and 2). All

MDR isolates were able to form biofilm. Five were

strong formers, six were moderate and only one with

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Location Kiosk Code No. of Sample No. of Isolate No. of Positive Isolate (%)

Kajang

R-1 3 8 2 (25.0)

R-2 3 7 2 (28.5)

R-3 3 10 2 (20.0)

R-4 3 12 2 (16.7)

R-5 3 8 1 (12.5)

South City

R-6 3 6 2 (33.3)

R-7 3 5 2 (40.0)

R-8 3 12 2 (16.7)

R-9 3 11 2 (18.2)

R-10 3 12 3 (25.0)

One South

R-11 3 6 1 (16.7)

R-12 3 10 3 (30.0)

R-13 3 9 3 (33.3)

R-14 3 6 1 (16.7)

R-15 3 6 1 (16.7)

Kuala

Lumpur

R-16 3 2 1 (50.0)

R-17 3 9 2 (22.2)

R-18 3 5 1 (22.2)

R-19 3 8 2 (25.0)

R-20 3 4 1 (25.0)

Total  60 156 36 (23.1)

Table 1 Detection of S. aureus from shawarma sandwich from kiosks (n = 20) in Klang Valley, Malaysia.

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Class of Antibiotics Antibiotic (ug)* Resistant (%) Intermediate (%) Susceptible (%)

 AMP 25 (69.4) - 11 (30.5)

Penicillin P 25 (69.4) - 11 (30.5)

Fluoroquinolone CIP 17 (47.2) - 19 (52.7)

 CN 1 (2.8) - 35 (97.2)

Aminoglycosides K 8 (22.2) 6 (16.7) 22 (61.1)

Cephem KF 1 (2.8) - 35 (97.2)

Tetracycline TE 12 (33.3) - 24 (66.7)

Folate pathway

inhibitor

W 2 (5.6) - 34 (94.4)

SXT 1 (2.8) - 35 (97.2)

Phenicol C - - 36 (100)

Nitrofurantoin F - - 36 (100)

Table 2. Antibiotic resistance profile of S. aureus isolates obtained from shawarma sandwiches (n = 60).

* AMP = ampicillin, P = penicillin G, CIP = ciprofloxacin, CN = gentamicin, K = kanamycin, KF = cephalothin, TE =

tetracycline, W = trimethoprim, SXT = trimethoprim-sulfamethoxazole, C = chloramphenicol, F = nitrofurantoin.

Isolate Antibiotic resistant profile* No. of antibiotics** MAR Index***

S1.1 P AMP W 3(2) 0.27

S1.2 P AMP 2(1) 0.18

S2.2 CIP 1(1) 0.09

S2.1 CIP 1(1) 0.09

S3.1 P AMP CIP 3(2) 0.27

S3.2 P AMP CIP TE 4(3) 0.36

S4.1 P AMP CIP TE K 5(4) 0.45

S4.2 P AMP CIP TE K 5(4) 0.45

S5.1 - - -

S6.1 P AMP CIP KF CN 5(4) 0.45

S6.2 P AMP 2(1) 0.18

S7.1 P AMP 2(1) 0.18

S7.2 P AMP 2(1) 0.18

S8.1 - - -

S8.2 P AMP CIP TE K 5(4) 0.45

S9.1 - - -

S9.2 P AMP CIP TE K 5(4) 0.45

S10.1 P AMP TE K 4(3) 0.36

S10.2 P AMP CIP TE K 5(4) 0.45

S10.3 P AMP 2(1) 0.18

S11.1 - - -

S12.1 - - -

S12.2 - - -

S12.3 P AMP CIP 3(2) 0.27

S13.1 P AMP 2(1) 0.18

S13.2 P AMP TE K 4(3) 0.36

S13.3 P AMP CIP TE 4(3) 0.36

S14.1 P AMP CIP 3(2) 0.27

S15.1 P AMP 2(1) 0.18

S16.1 - - -

S17.1 P AMP CIP 3(2) 0.27

S17.3 P AMP TE 3(2) 0.27

S18.3 CIP SXT W TE K 5(4) 0.45

S19.1 P AMP CIP TE K 5(4) 0.45

S19.3 CIP 1(1) 0.09

S20.2 P AMP 2(1) 0.18

Table 3. Multiple antimicrobial resistance (MAR) index value of S. aureus isolates.

AMP = ampicillin, P = penicillin G, CIP = ciprofloxacin, CN = gentamicin, K = kanamycin, KF = cephalothin, TE =

tetracycline, W = trimethoprim, SXT = trimethoprim-sulfamethoxazole, C = chloramphenicol, F = nitrofurantoin.

**The number of antibiotics to which each isolate was resistant. The number in the parenthesis indicates the total number of the

classes to which the antibiotics belong.

***Multiple antimicrobial resistances.

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weak ability. Additionally, all the sea gene-carrying

MDR isolates were strong biofilm formers. Moreover,

shawarma samples that contained MDR isolates which

carried sea genes and with strong biofilm formation

ability were detected from four kiosks; namely R-3, R-4,

R-8 and R-10.

4. Discussion

The occurrence of S. aureus in shawarma sandwich

in the current study (60%) was relatively higher than

those study reported for various Ready-to-Eat (RTE)

foods in Malaysia (Shafizi et al., 2016), China (Song et

al., 2015; Yang et al., 2016), Trinidad (Syne et al.,

2013), and Bangladesh (Islam et al., 2019), which

generally had the occurrence of S. aureus. In Sri Lanka,

32.0% S. aureus was found in shawarma and ham

(Wimalasekara, 2016).

In this study, the occurrence of S. aureus in

shawarma samples may be due to three main factors;

namely insufficient cooking of chicken meat,

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Kiosk code Isolate MDR profile *
sea genes sed genes Biofilm formation

** ** ***

R-3 SA3.2
3 class of antibiotic

+ nd +++
(a, b, e)

R-4

SA4.1
4 class of antibiotic

nd nd +++
(a, b, c, e)

SA4.2
4 class of antibiotic

+ nd +++
a, b, c, e

R-6 SA6.1
4 class of antibiotic

nd nd ++
a, b, c*, d

R-8 SA8.2
4 class of antibiotic

+ nd +++
a, b, c, e

R-9 SA9.2
4 class of antibiotic

nd nd ++
a, b, c, e

R-10

SA10.1
3 class of antibiotic

+ nd +++
a, c, e

SA10.2
4 class of antibiotic

nd nd ++
a, b, c, e

R-13

SA13.2
3 class of antibiotic

nd nd +
a, c, e

SA13.3
3 class of antibiotic

nd nd ++
a, b, e

R-18 SA18.3
4 class of antibiotic

nd nd ++
b, c, e, f

R-19 SA19.1
4 class of antibiotic

nd nd ++
a, b, c, e

Table 4. Characteristic of MDR isolates of S. aureus, the presence of sea and sed genes and their biofilm formation potential

* multi-drug resistance pattern based on antibiotic class; a = Penicillin, b = Fluoroquinolone, c = Aminoglycosides, d = Cephem,

e = Tetracycline, f = Folate pathway inhibitor

** genes detected = +; genes not detected = nd

*** biofilm formation; strong = +++; moderate = ++; weak = +

Figure 1. Visualized PCR products of Staphylococcal

Enterotoxin A (sea). Lane 1-7= Positive sample (SEA), Lane

8 = Positive control of S. aureus ATCC 13565, Lane 9 =

Negative control, Lane M =100 bp DNA marker

Figure 2. Visualized PCR products of Staphylococcal

Enterotoxin D (sed). Lane 2-9= Negative sample (SED), Lane

1= Positive control of S. aureus ATCC 13565, Lane 10=

Negative control, Lane M= 100 bp DNA marker

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contamination by raw ingredients and poor hygiene. Raw

meat of chicken has been reported contaminated with S.

aureus (Vazgecer et al., 2004). S. aureus was also found

isolated from 75% chicken and 52% turkey retail meats

(Tang et al., 2017). It was observed during this study that

piles of chicken meat were roasted on a rotating skewer,

with the outer layer of meat cooked properly, while the

inner part within the pile of meat remains raw and juicy.

During serving, the outer part of the meat block was

shaved, leaving the remaining block of meat to remain

heated on the skewer. Based on this cooking method,

shawarma samples may contain S. aureus cells that

survived the method of cooking as roasting chicken meat

block on the rotated skewer may cause insufficient or

inconsistent cooking of the chicken meat.

Moreover, raw ingredients added to the shawarma;

tahini sauce (from the oil of sesame seeds) and

vegetables could also be possible sources of

contaminants. S. aureus was found in vegetables such as

cabbage, tomato and lettuce, all were found to have high

contamination in ready-to-eat foods including shawarma

(Okafor et al., 2003; Meldrum et al., 2009; Sospedra et

al., 2013; Faour-Klingbeil et al., 2016). These vegetables

may be contaminated with soil that is possibly

transmitted to the hands of food handlers, and

consequently to the shawarma sandwich (Okafor et al.,

2003; Vazgecer et al., 2004; Harakeh et al., 2005;

Meldrum et al., 2009; Odu and Akano, 2012).

Food handlers are the key players that prevent food

contamination during food preparation by preventing

cross-contamination and by using proper cooking

methods and storage of cooked food (Soon et al., 2011).

This is also supported by random observation by the

authors during sample collection in this study, where the

majority of the workers in the kiosks did not wear gloves

during shawarma preparation and serving. Consequently,

the S. aureus occurrence in shawarma sandwiches is

likely a result of contamination by the food handlers due

to their poor hygiene practices. Thus, the contamination

of shawarma could be heavily related to the method of

preparation, as S. aureus is transmitted through sneezing,

coughing, and wounds in the hands of food handlers

(Odu and Akano, 2012; Abdalhamid et al., 2013; Nimri

et al., 2014).

In this study, 80.6% of S. aureus isolates were

resistant to at least one antimicrobial, with the

substantially higher resistance to the Penicillin class of

antibiotic; ampicillin (69.4%) and penicillin (69.4%).

Similarly, Geidam et al. (2012) found that more than

50% of the S. aureus isolated from the skin of poultry in

Malaysia exhibited resistance to penicillin and

ampicillin. Similar resistance to penicillin (53.3%) and

ampicillin (72.30%) was observed in S. aureus isolated

from the food handler’s hands in Malaysia (Tan et al.,

2014). This result is nearly analogous to that of

Arfatahery et al. (2016), who found that all the strains of

S. aureus isolated from shrimp and fish showed

resistance to penicillin and ampicillin. S. aureus resistant

to penicillin (99.3%) was also reported in a study

investigating dairy products in north-western Greece

(Papadopoulos et al., 2018). These results could be due

to the fact that antibiotics have been used increasingly

for the treatment of bacterial diseases in humans and

animals. Several antibiotics, especially for penicillin are

generally applied in veterinary medicine (Cháfer-Pericás

et al., 2010).

The resistance of the microorganisms to three or

more classes of antibiotics considered as multidrug-

resistant (MDR) (Gibbs et al., 2006; Waters et al., 2011).

In this study, twelve isolates were MDR and six of them

demonstrated MDR pattern that was dominated by four

antibiotic classes (penicillin, fluoroquinolone,

aminoglycosides and tetracycline). Multidrug-resistant

bacteria are now increasingly common because of the

indiscriminate use of antibiotics.

Chickens are possible sources of MDR bacteria

because various antibiotics are used to treat infections in

chickens and to promote their growth; hence, making it

possible for 100% of isolates from chicken in Oman to

exhibit multidrug resistance (Al-Bahry et al., 2007; Al-

Bahry et al., 2015) as compared to an earlier study

conducted in Oman in 1986 that reported only 30% of

the isolates being MDR (Al-Bahry, 1999). Multiple drug

resistance proves difficult to overcome especially by the

commonly used antibiotics and this intensifies the ability

of the bacteria to establish themselves in their hosts

leading to the production of more virulent factors that

enhance their survival (Tan et al., 2014; Frieri et al.,

2017).

The Emergence of multiple drug resistance among

bacteria from food handlers, food premises and food

products has also been a global concern (Altalhi et al.,

2010; Ryu et al., 2012; Rasheed et al., 2014; Tan et al.,

2014; Melo et al., 2015) and could lead to food

poisoning outbreaks that in turn leads to economic loss

and loss of lives if people of compromised immunity are

involved.

We targeted two classical SEs (sea and sed) genes

and found that of the thirty-six S. aureus isolates that

were resistant to at least one antibiotic, seven (19.4%)

harbored the sea genes. No sed gene was found in any of

the isolates tested. The sea gene is the most commonly

reported in contaminated foods and also in

staphylococcus-related food poisoning cases worldwide

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(Argudín et al., 2010; Gholamzad et al., 2015). A study

by Kérouanton et al. (2007) in France on S. aureus

linked to food poisoning outbreaks also revealed the

highest prevalence of SEA alone and in combination

with SED but relatively low percentages 13.9% and

6.7% respectively. The presence of S. aureus toxins in

foods is an indication for the potential to cause food

intoxication thereby leading to food poisoning outbreak.

Several studies involving the detection of S. aureus and

its enterotoxins from foods and food products have been

conducted worldwide. These include the presence of

enterotoxigenic S. aureus in ready-to-eat foods in Korea

(Kim et al., 2011), retail aquatic food products in China

(Rong et al., 2017), goat milk in China (Xing et al.,

2016), from food poisoning clone of S. aureus in Japan

(Sato'o et al., 2014). This study is the first study in

Malaysia and therefore there are not sufficient data to

make comparisons.

Although the production of sea genes was not

investigated in our study, the presence of sea genes

carrying S. aureus isolates in shawarma samples could

be of concern in terms of enterotoxin production. A

study on retail foods in China (Wang et al., 2017)

reported that more than 90% of the sea gene carrying S.

aureus isolates have produced enterotoxins.  Considering

these MDR isolates which also carry sea gene may enter

the food chain, and may be transferred to consumers,

their ability to form biofilm was established to evaluate

their persistence in the food environment. Our study

demonstrated that five of the 12 MDR isolates were

strong biofilm formers, and four of these MDR isolates

harboured sea genes.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgement

The authors would like to thank all the staffs from

the Faculty of Food Science and Technology who have

been involved throughout the study for all support and

assistance.

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