DOI: https://doi.org/10.52973/rcfcv-e32171

Received: 23/05/2022 Accepted: 23/07/2022 Published: 14/10/2022

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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32171, 1 - 11

ABSTRACT

In Turkish cuisine, ready–to–eat vegetable salads (REVS) served

with pide/lahmacun, kebab types, and tantuni from animal source

in meat restaurants were evaluated since they have the potential to

carry risks in terms of Public Health. The microbiological properties

of REVS were investigated using agar plate method. Antimicrobial

resistance of foodborne pathogens including Escherichia coli and

Staphylococcus aureus was tested using Kirby–Bauer disc diffusion

method. Moreover, the presence of important enteric viruses

was detected by Polymerase Chain Reaction (PCR). The number

of total aerobic bacteria, coliform bacteria, yeast and molds and,

Staphylococcus and Micrococcus spp. ranged from less than 1 to 6.40,

1 to 6.26, less than 1–5.82 and less than 1–5.66 log

colony forming

units·grams

-1

(CFU·g

–1

) in REVS samples, respectively. None of the

REVS tested in this study contained Salmonella spp., whereas E.

coli and S. aureus were isolated in 38.1% (16/42) and 2.4% (1/42),

respectively. S. aureus was resistant to gentamicin, kanamycin,

aztreonam, and ciprooxacin in the disc diffusion assay, however,

it was not harboring the mecA gene. E. coli strains (n=16) were resistant

(100%) to aminoglycoside antibiotics and 35.7% (6/16) of the isolates

were extended spectrum beta lactamase (ESBL) producing. bla

TEM

and

bla

CTXM8/25

were detected in two isolates, whereas one isolate carried

bla

CTXM–1

and bla

TEM

together by PCR. Of the REVS, two were evaluated

as positive for rotavirus (4.8%), six for hepatitis A (14%), and hepatitis

E virus (14%). These results indicate the high microorganism load,

presence of ESBL E. coli, and viral enteric pathogens in REVS, hence

it is important to perform routine hygiene practices.

Key words: Microbial; ESBL; E. coli; viruses; ready–to–eat salad

RESUMEN

Ensaladas de verduras listas–para–comer (EVLC) que se sirven con pita/

lahmacun, tipos de kebab y tantuni de origen animal en los asadores

de la cocina turca, ya que tienen el potencial de conllevar riesgos

en términos de salud pública fueron evaluadas. Se investigaron las

propiedades microbiológicas de REVS utilizando el método de placa de

agar. La resistencia a los antimicrobianos de los patógenos transmitidos

por los alimentos, incluidos Escherichia coli y Staphylococcus aureus, se

probó mediante método de difusión por disco de Kirby–Bauer. Además,

se detectó la presencia de importantes virus entéricos por Reacción

en Cadena de la Polimerasa (RCP). El número de bacterias aeróbicas

totales, bacterias coliformes, levaduras y mohos y Staphylococcus y

Micrococcus spp. varió de menos de 1 a 6,40; 1 a 6,26; menos de 1 a 5,82

y de menos de 1 a 5,66 log

Unidades Formadoras de Colonias·gramos

-1

(UFC·g

–1

) en muestras EVLC, respectivamente. Ninguna muestra de

EVLC analizadas en este estudio contenía Salmonella spp., mientras

que E. coli y S. aureus se aislaron en el 38,1% (16/42) y el 2,4% (1/42),

respectivamente. S. aureus fue resistente a la gentamicina, la

kanamicina, el aztreonam y la ciprooxacina en el ensayo de difusión

en disco; sin embargo, no albergaba el gen mecA. Las cepas de E. coli

(n=16) fueron resistentes (100%) a los antibióticos aminoglucósidos

y el 35,7% (6/16) de los aislamientos produjeron beta lactamasa de

espectro extendido (BLEE). bla

TEM

y bla

CTXM8/25

se detectaron en dos

aislados, mientras que un aislado portaba bla

CTXM–1

y bla

TEM

juntos

mediante RCP. De los EVLC, dos fueron evaluados como positivos

para rotavirus (5%), seis para hepatitis A (14%), y virus de la hepatitis

E (14%). Estos resultados indican la alta carga de microorganismos,

presencia de ESBL E. coli y patógenos virales entéricos en REVS, por

lo que es importante realizar prácticas de higiene de rutina.

Palabras clave: Microbiano; BLEE; E. coli; virus; ensalada lista para

comer

Microbiological quality of ready–to–eat vegetables salads served at meat

restaurants under the COVID-19 in Turkey

Calidad microbiológica de ensaladas de verduras listas para comer servidas en restaurantes

de carne durante la pandemia de COVID-19 en Turquia

Alper Baran

* , Mehmet Cemal Adigüzel

and Hakan Aydin

Atatürk University, Vocational School of Technical Sciences, Department of Food Quality Control and Analysis. Erzurum, Turkey.

Atatürk University, Faculty of Veterinary Medicine, Department of Microbiology. Erzurum, Turkey.

Atatürk University, Faculty of Veterinary Medicine, Department of Virology. Erzurum, Turkey.

*Email: alper.baran@atauni.edu.tr

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INTRODUCTION

Ready–to–eat (RTE) foods are those consumed without any

additional processing or preparation, which may be industrially and/

or conventionally processed, packaged or unpackaged [37]. Dietary

preferences of individuals due to rapid urbanization and socio–

economic transformations of their current lifestyles have shifted

towards ready–made meals [53].

Research and Markets [30] data showed that the demand for RTE

foods has increased rapidly, especially among consumers who do

not like to cook during the COVID–19 pandemic. In addition, it was

stated that, in connection with the increasing coronavirus cases,

restaurants forced consumers to cook at home, causing an increase

in the consumption of ready–made meals in the medium term.

On the other hand, the increasing consumption of RTE foods globally

has been signicantly associated with various outbreaks of foodborne

infections and poisoning. Especially RTE vegetable salads (REVS) can

be primarily contaminated by animals, soil, irrigation water, fertilizers,

and others. Moreover, cutting and slicing raw vegetables in restaurants

can cause the nutrients in the plants to be released and thus accelerate

microbial development [39]. Also, cross–contamination from staff

working in restaurants, tools/equipment used, contaminated water, as

well as storage in inappropriate conditions are important risk factors

affecting the nal microbiological quality of the product.

Therefore, REVS may carry microbial risks under the inuence

of more than one unsuitable factors [13, 33]. This situation can lead

to the emergence of pathogens such as Staphylococcus aureus,

Salmonella spp., Escherichia coli, Campylobacter jejuni, Clostridium

perfringens, and Listeria monocytogenes among others, as well as an

increased in total aerobic and spoilage bacteria, yeasts, and molds

in REVS [2].

On the other hand, a high microorganism load can be contained

multi–resistant bacteria of global health concern nowadays [48]. The

increasing presence of extended spectrum beta lactamase (ESBL)

producing bacteria from RTE foods, which is among the challenges

of antibiotic resistance, is another important risk for Human Health

due to its epidemiological importance [26]. In addition, it has been

reported that REVS are reservoirs not only for bacterial pathogens,

but also for enteric viruses such as norovirus (NoV), hepatitis A (HAV),

hepatitis E (HEV), and rotavirus (RV) that may lead to an epidemic [48].

The increasing interest in the demand for RTE foods in Turkey

has been in parallel with the developments in the World, especially

during the COVID 19 pandemic. RTE foods in which meat is used as

raw material such as pide/lahmacun, kebab types, and tantuni are

notably in high demand [30]. The demand to reach hygienic foods by

consumers is also for RTE foods, just like all other foods.

In some studies, conducted in Turkey, the microbiological quality of

RTE foods was investigated and it was reported the presence of some

foodborne pathogens including L. monocytogenes, Salmonella spp.

and Norovirus [3, 28, 51, 58, 67]. However, it is noteworthy that in these

studies, there was no comprehensive perspective on REVS in meat

restaurants. Therefore, it is thought that ignoring the microbiological

quality of the REVS may be an important risk in terms of Public Health.

Moreover, to the best of the knowledge, there is no adequate data

on the some microbiological in REVS from Eastern Turkey. Thus,

this study aimed to investigate some microbiological properties,

antimicrobial resistance of foodborne pathogens including E. coli

and S. aureus and the presence of enteric viruses in REVS in Turkey.

MATERIALS AND METHODS

Samples

In this study, REVS samples were taken aseptically from 42 meat

restaurants operating in Erzurum Region in Eastern Turkey. One

sample of REVS served with RTE foods was collected from each

restaurant. The samples were transferred to the on–ice packs in an

insulated cooler. Samples were analyzed much less than 2 hours (h)

after collection.

TABLE I presents information about the characteristics of REVS and

restaurants. Information on the conditions under which most of the

samples used in the study were stored was not given by the restaurant

staff. REVS samples, which did not contain any additives, dressing

and gravy were placed directly on a suitable packaging material and

made ready for service. Since all REVS collected as a sample were

washed, grated, and ready for direct consumption, it was named RTE.

TABLE I

Characteristics of collected REVS and restaurants

Symbol Ingredients of Vegetable Origin Packaging Method Restaurant type

Carrot and lettuce Polyethylene Döner kebab (Chicken)

Parsley, salad, and tomatoes Polystyrene foam Pide–Lahmacun

Carrot, parsley, and tomatoes Polyethylene Döner kebab (Chicken and beef)

Carrot and lettuce Polystyrene foam Döner kebab (Chicken and beef)

Carrot, lettuce, and purple cabbage Polyethylene Döner kebab (Chicken and beef)

Carrot, lettuce, purple cabbage, and tomatoes Polystyrene foam Shish kebab

Lettuce and tomatoes Polyethylene Döner kebab (Chicken)

Lettuce and tomatoes Polyethylene Döner kebab (Chicken and beef)

Carrot, lettuce, and purple cabbage Polystyrene foam Pide–Lahmacun and Döner kebab (Beef)

E10

Lettuce Polyethylene Tantuni

E11

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun

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Symbol Ingredients of Vegetable Origin Packaging Method Restaurant type

E12

Carrot, lettuce, and purple cabbage Polyethylene Döner kebab (Chicken)

E13

Lettuce and tomatoes Polyethylene Döner kebab (Chicken)

E14

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun

E15

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun, Shish kebab, Döner kebab (Beef)

E16

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun, Shish kebab

E17

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun

E18

Lettuce and tomatoes Polyethylene Döner kebab (Chicken)

E19

Lettuce and tomatoes Polyethylene Döner kebab (Chicken)

E20

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun, Shish kebab, Döner kebab (Beef)

E21

Carrot and lettuce Plastic bag Döner kebab (Chicken)

E22

Carrot and lettuce Polyethylene Döner kebab (Chicken)

E23

Carrot Polyethylene Döner kebab (Chicken)

E24

Carrot, lettuce, and purple cabbage Polyethylene Döner kebab (Chicken and beef)

E25

Carrot, lettuce, and tomatoes Polyethylene Döner kebab (Chicken and beef)

E26

Carrot and lettuce Polyethylene Döner kebab (Chicken)

E27

Carrot and lettuce Polyethylene Döner kebab (Chicken)

E28

Carrot, lettuce, and tomatoes Polystyrene foam Shish kebab

E29

Carrot and lettuce Polystyrene foam Döner kebab (Chicken)

E30

Lettuce Polyethylene Döner kebab (Chicken)

E31

Lettuce Polystyrene foam Döner kebab (Chicken and beef)

E32

Lettuce Polyethylene Döner kebab (Chicken)

E33

Lettuce Plastic bag Döner kebab (Chicken)

E34

Carrot, lettuce, and purple cabbage Polyethylene Pide–Lahmacun, Shish kebab

E35

Carrot, lettuce, and purple cabbage Cardboard bag Pide–Lahmacun, Shish kebab, Döner kebab (Beef)

E36

Carrot, lettuce, and purple cabbage Polyethylene Döner kebab (Chicken)

E37

Lettuce, salad, and tomatoes Polystyrene foam Giblet shish kebab

E38

Carrot, lettuce, purple cabbage, and tomatoes Polyethylene Tantuni

E39

Carrot, lettuce, purple cabbage, and tomatoes Polyethylene Pide–Lahmacun, Shish kebab, Döner kebab (Beef)

E40

Lettuce and tomatoes Plastic bag Döner kebab (Chicken and beef)

E41

Lettuce and tomatoes Polyethylene Döner kebab (Chicken and beef)

E42

Carrot, parsley, and purple cabbage Polystyrene foam Döner kebab (Chicken)

TABLE I (cont...)

Characteristics of collected REVS and restaurants

Microbiological analysis

A 10 grams (g) sample was transferred to a ltered stomacher bag

and 90 milliliters (mL) of sterile physiological saline solution (0.85%

NaCI) was added. The mixture was homogenized in a stomacher

(Masticator® Mixer, Basic, Neutec Group Inc., USA) for one minute

(min). From this homogenate, 10–fold serial dilutions were prepared

with sterile physiological saline solution, and 0.1 mL various dilution

levels were spread–plated onto appropriate media and incubated in

a laboratory oven (BINDER, Series ED, Binder GmbH, Germany) at

appropriate condition (TABLE II). The bacterial colonies were counted

and converted in colony forming units (CFU). CFU was calculated

following formula:

CFUg Number of colonies

Volume actually plated

Totaldilution

All counts were reported as log

CFU·g

–1

[11].

The samples were analyzed for the presence of foodborne

pathogens commonly isolated from REVS, i.e., E. coli, Salmonellaspp.,

and S. aureus. To isolate E. coli, Salmonellaspp., and S.aureus, 25g of

each sample were aseptically weighed (Shimadzu, ATX 224, Shimadzu

Scientic Instruments, Japan) transferred to sterile ltered plastic

bags, and homogenized using masticator with 225 mL buffered

peptone water (BPW) (Merck, Germany) for 2 minutes (min). Then

this homogenate was kept for 60 min at room temperature [7]. For

Salmonellaspp. isolation, the homogenate was incubated (BINDER,

Microbiological quality of salads in meat restaurants in Turkey / Baran et al. __________________________________________________________

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Series ED, Binder GmbH, Germany) for 24 ± 2 h at 35°C followed by a 0.1

mL mixture transferred to 10 mL Rappaport–Vassiliadis (RV) medium

and another 1 mL mixture to 10 mL Muller–Kauffmann Tetrathionate–

Novobiocin Broth (MKTTN) and they were incubated (BINDER, Series

ED, Binder GmbH, Germany) at 37°C for 24 h.

A loop–full culture of each broth was streaked onto Xylose Lysine

Desoxycholate (XLD) agar and Xylose Lysine Tergitol 4 (XLT4) agar and

incubated at 37°C for 24 h. Two or more colonies of showing typical

Salmonella colony morphology were picked from each selective agar

plate after incubation [11]. To E. coli isolation, a loop–full of pre–

enriched sample was streaked onto Tryptone Bile X–glucuronide (TBX)

agar and incubated at 37°C for 24 h. The blue–green colored suspicious

E. coli colonies grown in TBX were subcultured on the Mueller Hinton

agar (MHA) (HiMedia, Mumbai, India) plate for biochemical and

molecular analysis [10]. A loop–full of homogenate was passaged

on BP Agar supplemented with egg yolk tellurite emulsion to isolate

S. aureus and incubated at 37°C for 24 h. The typical transparent

zone around the colony as a result of lipolysis/proteolysis, black

colonies due to the reduction of tellurite to tellurium were evaluated

as suspicious S. aureus, and subcultured for further analysis as

mentioned for E. coli above [27].

Antimicrobial susceptibility testing

Kirby–Bauer disk diffusion technique on MHA (HiMedia) was

used for antimicrobial susceptibility testing against to a set of 12

different commercially available antibiotic disks (HiMedia, Mumbai,

India) including Meropenem (10 micrograms – µg-), Chloramphenicol

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

Ciprooxacin (5 µg), Sulfamethoxazole/Trimethoprim (25 µg), Cefepime

(30 µg), Cefpodoxime (10 µg), Cefoxitin (30 µg), and Aztreonam (30µg).

The growth inhibition zones were measured and interpreted as

sensitive or resistant as recommended by the guidelines of Clinical

& Laboratory Standards Institute (CLSI) [64]. Resistance to at least one

agent in three or more antimicrobial groups was dened as multi–drug

resistance. E. coli ATCC 25922 was used as control strain.

Phenotypic and genotypic detection of ESBL E. coli and Methicillin–

resistant S. aureus

The presence of ESBL phenotype was determined by the double–

disc synergy test (DDST) using Cefotaxime (CTX) and Ceftazidime (CAZ)

alone and in combination with Clavulanic acid as recommended by the

CLSI [64]. For this purpose, CAZ (30 μg), CTX (30 μg), CAZ–Clavulanic

acid (30/10 μg), and CTX–Clavulanic acid (30/10 μg) discs were placed

on MHA. After 16–18 h of incubation at 35°C, ≥ 5 millimeters (mm)

increase in zone diameter of CAZ/Clavulanic acid disc and CAZ disc

alone, and/or ≥ 5 mm increase in the zone diameter of CTX/Clavulanic

acid disc and CTX disc alone were considered as ESBL positive.

The genotypic assay was made by using genomic deoxyribonucleic

acid (DNA) obtained from phenotypic ESBL positive isolates by

boiling method as a template. Briey, 100 microliters (μL) of Tris–

Ethylenediaminetetraacetic acid (EDTA) buffer solution (pH 8.0)

containing a few colonies were boiled in a dry heating block (TDB–100,

Biosan, Latvia) for 10 min. At the end of the heating, the samples were

cooled on ice and centrifuged (MIKRO 220R, Hettich, Germany) at

10.000 x G force – G – for 15 seconds (s). The supernatant was used

as template in polymerase chain reaction (PCR) [10], components

were provided by Vivantis Technologies (Subang Jaya, Malaysia).

Primers used in this study were obtained from Metabion

International AG (Planegg‐Martinsried, Germany). The isolates that

were ESBL–positive by double disc synergy test (DDST) were subjected

to amplication by PCR method using TEM, SHV, CTX–M–1, CTX–M–2,

CTX–M–8/25, and CTX–M–9 group primers, as previously reported by

Le et al. [35]. The PCR amplications were performed in a total volume

of 15 μL solution containing 2 μL of template DNA, 1X PCR buffer

(Sigma), 0.25 milimolar – mM – MgCl

(Sigma), 200 micromole – μM –

(each) dNTP (Sigma), 10 picomoles of each primer, 1.25 Units – U – of

Taq polymerase (Sigma). The reaction conditions for amplication

of DNA were 95°C for 5 min, 25 cycles of 95°C for 30 s, 60°C for 90 s

and 72°C for 90 s, and 68°C for 10 min.

Oxacillin disc (1µg; Oxoid, Cambridge, United Kingdom–UK) diffusion

assay was performed for evaluation of Methicillin–resistant S. aureus

(MRSA) following CLSI guidelines for interpretation of the results

and using S. aureus ATCC 29213 as a CLSI negative control. PCR was

performed for the conrmation of S. aureus isolates with femA gene

and presence of Methicillin–resistant gene mecA [23]. PCR reaction

mixture was prepared the same mentioned above and the reaction

condition was initial denaturation at 95°C for 5 min, 35 cycles of

amplication (denaturation at 95°C for 2 min, annealing at 58°C for

1 min, extension at 72°C for 1 min), and nal extension at 72°C for 10

min in a thermal cycler.

Phylo–typing analysis

Phylo–typing groups were determined using the quadruplex

phylogroup assignment method for E. coli isolates, previously described

by Clermont [18]. PCRs were carried out using thermal cycler (BioRad,

USA) in a total volume of 25 μL containing 10 pmol of each three pair

of primers (Sigma, USA), 25 μM of dNTPs, 5 μL of template DNA, 2.5

μL of 10X Taq buffer [50 mM KCl, 10 mM Tris–HCl (pH 8.3)], 2 mM MgCl

and 2.5 U of Taq polymerase (Fermentas, USA). The PCR products

were separated by electrophoresis through 1.2% agarose gel in 1X TAE

buffer. DNA fragments were visualized by ethidium bromide staining

and photographed under ultraviolet light illumination (gelDoc

XR+

Gel Documentation System, BioRad,USA) [11].

TABLE II

Media and incubation conditions used for the

enumeration of microorganisms

Microorganisms

Incubation

Culture media

Time (h) Temp (ºC)

Total aerobic

mesophilic bacteria

72 35 Plate Count Agar (PCA)

Coliforms 24 35 Violet Red Bile Agar (VRBA)

Staphylococcus /

Micrococcus spp.

48 37

Baird Parker Agar (BPA)

(supplemented with egg

yolk tellurite emulsion)

Yeast and Mold 168 25

Rose Bengal

Chloramphenicol (RBC) Agar

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Viral ribonucleic acid (RNA) detection

PCR analysis was performed to investigate rotavirus (RV), hepatitis

E virus (HEV), hepatitis A virus (HAV) , and Norovirus (NoV), that could

be transmitted by the fecal–oral route. Primer sets used for each virus

are shown in TABLE III [9, 29, 46, 60]. Each of the REVS samples were

put 2 g in the tube and diluted with 1.5 mL of phosphate–buffered

saline (PBS). After vortexing, it was centrifuged at 770 G at 4°C for

5 min and 500 µL of supernatant from this suspension was used for

isolation of viral nucleic acid (VNA). VNA was extracted from RTE

salad samples by using the GF–1 VNA extraction kit (Vivantis, Malaysia)

according to the manufacturer’s instructions. A NA concentration

was measured using a NanoDrop spectrophotometer (NanoDrop

1000, Thermo Scientic, USA). The amplicons obtained from PCR

were visualized by gel–electrophoresis and positive results on the gel

were recorded for each virus. Positive control samples were viruses

that have been previously conrmed by sequence analysis [5, 9].

TABLE III

Rotavirus, Hepatitis E virus, Hepatitis A virus and

Norovirus PCR primer sequence used in this study

Primer Sequence 5’–3’ Length (bp)

Rotavirus

VP6 F

VP6 R

GACGGVGCRACTACATGGT

GTCCAATTCATNCCTGGTGG

379

Hepatitis A

Virus

VP1/P2B F1

VP1/P2B R1

VP1/P2B F2

VP1/P2B R2

GACAGATTCTACATTTGGATTGGT

CCATTTCAAGAGTCCACACACT

CTATTCAGATTGCAAATACAAT

AACTTCATTATTTCATGCTCCT

512

394

Hepatitis E

Virus

3156 F

3157 R

AATTATGCC(T)CAGTAC(T)CGG(A)GTTG

CCCTTA(G)TCC(T)TGCTGA(C)GCATTCTC

731

Norovirus

JV12 F

JV13 R

ATACCACTATGATGCAGATTA

TCATCATCACCATAGAAAGAG

327

Statistical analysis

The descriptive statistics were calculated for each parameter using

SPSS Software (IBM SPSS statistics 20, USA).

RESULTS AND DISCUSSION

Microbiological quality of REVS

Of a total of forty–two REVS samples were collected from meat

restaurants operating in the Erzurum Region in Eastern Turkey to

investigate the foodborne pathogens including bacteria and viruses

for their microbiological properties and antimicrobial resistance. The

total aerobic bacteria (TAB) in the tested REVS samples ranged from

less than 1 to 6.40 log

CFU·g

–1

(with an average of 4.72 log

CFU·g

–1

Although the lowest TAB count was detected in the samples #E14

and #E16, the highest value was in the #E24 (TABLE IV).

In addition, the highest yeasts and molds (5.00 log

CFU·g

–1

), coliform

(5.79 log

CFU·g

–1

), and Staphylococcus and Micrococcus bacteria (5.66

log

CFU·g

–1

) have been also detected in the sample #24. The count of

bacteria in the salad samples tested in this study results showed an

overall similar trend to each other. For example, when the TAB count

was detected as high, other parameters showed a high trend as much

as TAB (TABLE IV). A very few of the samples 3 out of 42 (7.14%) had

a TAB count greatest than 6 log

CFU·g

–1

, which categorizes them as

borderline, according to Health Protection Agency (HPA) guidelines [25].

It has been reported that TAB was ranged from 3.0 log

CFU·g

–1

to 6.7 log

CFU·g

–1

in RTE salads in Mexico city [15], however, it was

between 5.12 and 9.75 log

CFU·g

–1

(with an average of 7.73 log

CFU·g

–1

in REVS in Cyprus [65]. In addition, a high level of TAB (6.43 log

CFU·g

–1

-mean {3.50–8.39}) was reported in raw salad vegetables sold

in Lebanon [22]. Of note, these ndings were found to be considerably

higher than the present study. On the other hand, the high level of

yeast and molds (ranged between less than 1 to 6.26 log CFU g

–1

) were

detected in the REVS samples. In the previous studies, which were

consistent with the current study results, the yeast and mold were

reported 10

–10

log

CFU·g

–1

and 1.63 and 6.68 log

CFU·g

–1

from India

[44] and Mexico [27], respectively.

TABLE IV

Microbiological quality of REVS

Microbial Count (log

CFU·g

–1

) Presence (+/–)

Symbol TAB YM CO STA/MICR STA SAL EC HAV RV HEA NoV

4.60 3.30 3.83 1.30 – – + – – – –

4.48 3.53 2.48 1.85 – – – – – – –

5.51 6.26 5.06 1.30 – – – – – – –

4.78 4.23 4.65 1.48 – – + – – – –

4.90 5.06 4.40 2.20 – – + – – – –

5.20 5.43 5.25 1.00 – – + – – – –

5.04 4.46 4.74 1.30 – – – – – – –

5.97 5.45 4.88 1.30 – – – – – – –

5.45 5.08 5.03 1.00 – – – – – + –

E10

5.00 4.88 4.83 1.48 – – – – – – –

Microbiological quality of salads in meat restaurants in Turkey / Baran et al. __________________________________________________________

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Microbial Count (log

CFU·g

–1

) Presence (+/–)

Symbol TAB YM CO STA/MICR STA SAL EC HAV RV HEA NoV

E11

4.81 3.18 4.46 <1.00 – – – + – – –

E12

5.81 5.59 5.46 1.70 – – – – – – –

E13

4.90 4.86 4.89 1.30 – – – – – – –

E14

<1.00 3.48 3.70 <1.00 – – + – – – –

E15

5.00 5.09 5.06 1.48 – – – – – – –

E16

<1.00 3.00 <1.00 <1.00 – – + – – – –

E17

6.34 5.34 5.82 2.81 – – – – – – –

E18

4.00 4.82 4.11 1.48 – – + – – – –

E19

4.00 <1.00 3.30 <1.00 – – – – – – –

E20

4.48 3.70 3.60 2.64 – – – – – – –

E21

4.60 5.05 4.00 2.51 – – + – + + –

E22

5.81 5.09 4.58 2.82 – – + – – – –

E23

4.00 3.48 3.78 <1.00 – – – – – – –

E24

6.40 5.00 5.79 5.66 – – + – – + –

E25

4.30 4.40 3.78 <1.00 – – – – – – –

E26

4.60 3.90 4.78 2.04 – – – – – – –

E27

5.20 3.60 4.98 2.04 – – – – – – –

E28

5.32 5.00 4.68 1.90 – – – – – – –

E29

4.30 4.00 4.20 <1.00 – – – – – – –

E30

4.70 3.48 4.38 2.92 – – – – – + –

E31

4.30 4.58 3.78 1.70 – – + – – – –

E32

5.76 4.54 4.68 <1.00 – – – – – – –

E33

4.00 3.30 3.60 1.00 – – + – – – –

E34

4.00 4.11 5.10 1.78 – – + – – – –

E35

5.15 3.60 4.93 2.00 – – – – – + –

E36

5.04 4.61 4.67 1.60 – – – – – – –

E37

5.11 4.57 4.80 2.92 – – – + – – –

E38

4.78 3.48 4.40 2.53 – – – – – – –

E39

6.39 4.11 4.00 1.70 – – + + – + –

E40

4.95 4.08 4.78 2.38 + – – + – – –

E41

4.95 4.30 4.80 2.23 – – + + – – –

E42

4.30 4.23 4.08 2.70 – – + + +

– –

MinV

<1.00 <1.00 <1.00 <1.00

MaxV

6.40 6.26 5.82 5.66

Variance

1.56 1.04 0.93 1.25

1.25 1.02 0.96 1.12

0.19 0.16 0.15 0.17

Mean

4.72 4.27 4.38 1.62

*TAB: Total aerobic bacteria, STA/MICR:

Staphylococcus/Micrococcus, CO: Coliform bacteria, YM: yeasts and molds, SAL: Salmonella spp., STA: S. aureus,

EC:

E. coli, HEV: Hepatitis E, RV: Rotavirus, HAV: Hepatitis A, NoV: Norovirus; <1.00: below the detection level. The results are shown as log

CFU·g

–1

; (+)

presence of bacteria and viruses in 25 g and 2 g of the product, respectively, (–) absence of bacteria and viruses in 25 g and 2 g of the product, respectively

TABLE IV (cont...)

Microbiological quality of REVS

________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32171, 1 - 11

7 of 11

In this context, coliform bacteria (CO) were detected at levels

ranging from less than 1 to 5.82 log

CFU·g

–1

in all REVS samples.

Regardless of the sources, the number of CO was detected in all

salads served in Mexican restaurants with limits ranging from

5.4×10

to 1.7×10

log

CFU·g

–1

[27]. The high CO count identied in

the current study may be associated with poor hygiene practices

during the preparation of REVS. Apart from CO, the Staphylococcus

and Micrococcus spp. count (STA/MICR) was determined between

less than 1 and 5.66 log

CFU·g

–1

in the current study, indicating a

possible transmission from the food handlers. Risk factors that

play a role in the contamination of vegetables, such as unsafe water

sources for irrigation, inappropriate fertilizers or manures, access

to livestock wild animals in the eld, and unhygienic post–harvest

handling (unhygienic utensil, labor, handling, packaging material,

and improper/inadequate storage conditions) were indicated by

investigators in the previous studies [16, 38, 39, 47]. These results

showed a relatively high contamination rate detected in the salad

samples tested in this study, indicating that RTE salad serving in the

meat restaurant is a risk factor for the transmission of food–borne

diseases in humans [12].

Presence of E. coli, S. aureus and Salmonella spp. in REVS

Although the HPA guidelines indicated greater than or equal to 10

for E. coli, greater than or equal to 10

for S. aureus, and presence in

25 g for Salmonella spp. These are evaluated as an unsatisfactory

product [25], Turkish Food Codex [59] has ruled that RTE foods should

not contain E. coli (less than 10

), Salmonella spp., L. monocytogenes,

and staphylococcal enterotoxins. None of the REVS tested in this

study contained Salmonella spp., whereas E. coli and S. aureus were

isolated in 38.1% (16/42) and 2.4% (1/42), respectively, however, not

using the colony counting method. Hence, it is impossible to evaluate

the current study results according to HPA guidelines and/or the

Turkish Food Codex (TFC) [59]. In contrast to current study result,

it has been reported that a higher level of S. aureus (12 and 13.02%)

was detected in ready–to–eat salads in Turkey [8, 41].

Considered by food vendors as an indicator of fecal contamination

and improper hygiene practices, E. coli can cause gastroenteritis

and diarrhea in humans when taken with contaminated food [2]. The

prevalence of E. coli was found to be 38.10% (16/32) in REVS samples

analyzed in this study. This result was lower than in studies done in

other Countries: 96.7% in Ghana [2]; 94% in Cote d’ivoire [19]; 83.2%

in Mexico [27]; 64% in Argentina [43]. But it was higher than some

Countries: 20% in United Arab Emirates [6] and 16.7% in Spain [1].

The prevalence of E. coli contamination has displayed a signicant

variation between developed and developing countries [47]. For

example, studies in low–income Countries such as Pakistan,

Bangladesh, and India reported the prevalence of E. coli in raw vegetable

and ready–to–eat salad samples sold in retail markets ranged from 34

to 60% [4, 49, 55] whereas it was 3.1 and 4.0% in the USA and Turkey,

respectively [28, 36].

Phylo–typing of E. coli strains using quadruplex PCR displayed six

isolates in group A, four isolates in groups B1 and C, and one isolate

in groups E and F (TABLE V). Although most of E. coli isolates were

detected in the group with commensal strains (group A and B1) in the

current study, only one isolate was detected in the virulent group (F).

Despite the high STA/MICR count, S. aureus could only be detected

in one sample in the current study. As it is known, S. aureus is one of

the most important foodborne pathogens worldwide, some strains

of which can produce one or more toxins, mainly enterotoxin [64].

The prevalence of S. aureus in REVS observed in the present study

is comparable to similar studies [22, 40, 54].

The sources of contamination of Salmonella could be animal feces

as fertilizer, cultivation of the plants with wastewater, and personal

hygiene [40, 50, 52, 57]. Salmonella spp. was also isolated in fresh

vegetables by investigators in the previous studies [21, 52, 66] in

contrast to the present study ndings. Similarly, it has been reported

none of Salmonella spp. was detected from 45 REVS in Portugal [14].

This result suggests it might be no cross–contamination with Salmonella

spp. during the sample collection in the current study.

Antimicrobial resistance of the isolates from REVS

Only one S. aureus was isolated and conrmed by PCR (with femA

gene) in the current study, and the isolate was resistant to gentamicin,

kanamycin, aztreonam, and ciprooxacin in the disc diffusion assay. In

addition, the PCR analysis conducted for detection of the mecA gene

showed that the S. aureus strain isolated in the current study was not

harboring the mecA gene. On the other hand, all E. coli strains (n=16)

were resistant (100%) against aminoglycoside antibiotics (gentamicin

and kanamycin) tested in this study, even though they were susceptible

to meropenem in the disc diffusion assay (TABLE VI). The isolates

displayed a low level of resistance against chloramphenicol (6.25%),

tetracycline (18.75%), and ciprooxacin (25.00%). These data indicated

that antimicrobial–resistant E. coli strains from REVS salad samples

in the current study still remain moderately low–level resistant to anti–

Gram–negative drugs of importance for Human Medicine, suggesting

that these antibiotics could be non–effective in the future due to

the rising antimicrobial resistance. The double–disc synergy test

used for the detection of the phenotypic resistant ESBL producing

TABLE V

Phylogenetic groups and ESBL presence of E. coli

isolates from RTE vegetable salad samples

Isolate ID Phylogeny ESBL ESBL Genotype

E1 B1 –

E4 B1 –

E5 A –

E6 B1 –

E14 A –

E16 B1 + CTX–M–1, TEM

E18 C –

E21 A –

E22 A –

E24 C + –

E31 C –

E33 A –

E34 A + CTXM8/25

E39 E + –

E41 C + TEM

E42 F + –

Total 16/42 6/16

FIGURE 1. Rotavirus, Hepatitis E virus, and Hepatitis A virus positive

PCR amplicons

Microbiological quality of salads in meat restaurants in Turkey / Baran et al. __________________________________________________________

8 of 11

strains in the current study displayed that 35.7% (6/16) of the isolates

were ESBL producing. Molecular analysis of ESBL producing strains

showed that one strain harbored two different genes (bla

CTXM–1

and

bla

TEM

), whereas two isolates carried one gene (bla

TEM

and bla

CTXM8/25

To the best of this knowledge, this is the rst report of the presence

of ESBL producing E. coli in REVS in Turkey. The outbreaks due to the

multi–drug resistant bacteria on fresh vegetable products have been

reported around the world by researchers in previous studies [24,

31, 36, 52]. A study in Japan indicated that fresh vegetables served

as an important route of transmission of ESBL producer bacteria

to humans [61].

In terms of Public Health, antimicrobial resistant zoonotic

pathogens in foods pose a direct risk. Foods can be contaminated

with bacteria harboring antimicrobial resistance genes, antibiotics

using agricultural production, resistance genes of microorganisms

used as starters during food processing, and cross contamination.

Since raw foods are consumed without undergoing any other

processing, they carry a signicant risk of transferring antimicrobial

resistance to humans. Ultimately, transfer of antimicrobial resistance

genes between bacteria can also occur after ingestion by humans

[63]. Moreover, poor processing and preservation conditions lead

to the continued presence of damaged or stressed cells in food,

increasing the risk of bacteria carrying antimicrobial resistance

genes transmission [34].

Viral foodborne pathogens from REVS

The presence of HEV, RV, HAV and NoV was investigated in the

current study to reveal important viral infections to Public Health in

the REVS samples. VNA was detected in 14/42 salad samples tested

in the current study by PCR. Of these, two were evaluated as positive

for RV (5%), and six for HAV (14%) and HEV (14%). NoV could not be

found in any of the samples in the current study. Gel–electrophorese

images of VNA and control groups determined positive by PCR analysis

were given in FIG. 1. HEV, HAV, RV, and NoV are transmitted to humans

by food and environmental routes depending on the virus genotype,

environmental conditions, hygienic conditions, and the types of food

consumed [62].

TABLE VI

Antibiotic susceptibility pattern (Sensitive

and Resistance) of E. coli isolates

Antibiotic

Antibiotic susceptibility pattern

Sensitive Resistance

Meropenem

(10 µg)

16 (100%) 0

Chloramphenicol

(30 µg)

15 (93.75%) 1 (6.25%)

Gentamicin

(10 µg)

0 16 (100%)

Kanamycin

(30 µg)

0 16 (100%)

Tetracycline

(30 µg)

13 (81.25%) 3 (18.75%)

Ciprooxacin

(5 µg)

12 (75.00%) 4 (25.00%)

Sulfamethoxazole/Trimethoprim

(25 µg)

11 (68.75%) 5 (31.25%)

Cefepime

(30 µg)

14 (87.5%) 2 (12.50%)

Cefpodoxime

(10 µg)

9 (56.25%) 7 (43.75%)

Cefoxitin

(30 µg)

12 (75.00%) 4 (25.00%)

Aztreonam

(30 µg)

15 (93.75%) 1 (6.25)

In a study showed that in the total of 911 REVS samples from

supermarkets in Italy, the total prevalence of HAV and HEV was 1.9%

(18/911) and 0.6% (6/911), respectively, even though NoV could not be

detected in any of the samples [58]. The prevalence of HAV and HEV

was high in the samples tested in this study whereas NoV was not

detected. In contrast to the obtained study data, a low level of NoV

(2.90–3.75%) was reported in vegetables and fruits [42], indicating

the less frequent detection of NoV in REVS products. Hence, salads

are less frequently involved in foodborne viral outbreaks than other

foods; however, they may be contaminated with unsanitary food

workers or raw materials that have been contaminated [17]. Khan et

al. [32] reported that 29 vegetable samples collected from 13 different

locations of District Mardan in Pakistan, one was positive for HAV.

Shin et al. [56] reported that one sample was positive for HAV in

fresh vegetables and fruits from supermarkets in Mexico, 7/80 were

positive for HAV [42]. In another study, of the 70 vegetable samples

including 51 rst range raw vegetables and one fourth range REVS

from markets in Sicily,Italy, 1.4% for HEV [45]. The prevalence of RV

was found variable in vegetables: 13.75% (11/80) in Mexico [42]; 22%

(23/101) in Argentina [20]. To the best of the present knowledge, this

is the rst report from Turkey for HEV, HAV, and RV positivity in REVS,

suggesting REVS can be a reservoir for the important viral pathogens

and to be considered before consumption.

Viral contamination can occur at several points in the food

production chain. Because they do not have a chance to growth

outside of living cells, their presence in food can be explained by pre–

harvest contamination of vegetables or post–harvest contamination

from food processors. On the other hand, the fact that the food

handlers in the eld where the vegetables are harvested and the water

quality used in agricultural irrigation can affect the microbiological

properties of vegetables can also explain the high level of viral

contamination [58].

________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32171, 1 - 11

9 of 11

CONCLUSIONS

The assessment of microbiological contamination status in REVS

contributes to the identication of risks for government authorities

as well as to the assessment of consumer exposure. This is the rst

report of the presence of ESBL producing E. coli, HEV, HAV and RV

for REVS from Turkey. On the other hand, it can be predicted that

the high microorganism load detected in REVS samples and the

antimicrobial resistance of isolates may pose a threat to Public Health.

Considering that the lack of an effective surveillance system led to the

inability to identify the possible source of the epidemic, it was thought

that REVS could be a reservoir in this sense. Elimination or at least

mitigation of this current potential risk requires increasing the good

agricultural and manufacturing practices throughout the vegetable

production process from the farm to the retailer and restaurants.

Moreover, hazard analysis and critical control point (HACCP) strategies

need to be implemented and effectively supervised, especially at

the restaurant level.

Conict of Interest

The authors declare that they have no conicts of interest in the

research

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