An Adenosine Triphosphate Bioluminescence Method for Evaluating the Microbial Contamination of the Salad-Preparing Tables and Salad-Serving Dishes in Restaurants of Mashhad City, Iran

1 Department of Statistics and Epidemiology, School of Health, Mashhad University of Medical Sciences, Mashhad, Iran. 2 Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran. 3 Department of Environmental Health Engineering, School of Health, Mashhad University of Medical Sciences, Mashhad, Iran. 4 Environmental Health Expert of Mashhad Health Center No. 5, Mashhad University of Medical Sciences, Mashhad, Iran.


Introduction
To keep a crucial healthy diet, it is essential to consume fresh and worthy vegetables 1 . Previous studies indicated that vegetables may be contaminated by human pathogenic bacteria during the harvest and distribution processes 2 . In recent decades, due to the high costs of effluent treatment and deficiency of the freshwater resources, many reports showed irrigation of the cultivation lands using infectious effluent, raw sewage, and use of animal and human fertilizers 3 . Considering that no chemical treatments are carried out to reduce the microbial contamination in this field, vegetables can convey pathogenic bacteria that are transmittable through food 2 . Food-borne diseases are considered as the most important health problems in the world 4 . According to the Center for Disease Control and Prevention (CDC), about 41 million cases of disease and 3000 deaths occur a year due to food-borne illnesses in the United States 5 . According to the World Health Organization (WHO), food code commission, ready-to-use cooked foods, and all kinds of salads are classified in the high risk group of foods 6 . Vegetable salads are served raw, which makes them susceptible to microbial contamination during the preparation processes. Time and temperature, personal hygiene, and handling practices are among the most important parameters that affect this process. Hence, to prevent crosscontamination and transmission of food-borne pathogens, these parameters should be strictly considered 7 .
In most studies, microbial contamination of foods has been investigated, while the possibility of microbial contamination transmission at other levels, such as contamination of food preparation equipment and consumption should also be investigated 5 . The catering businesses were responsible for more than 59% of the outbreaks caused by food-borne diseases in Europe from 1993 to 1998. Similarly, food-borne diseases were responsible for 55% of different outbreaks in Spain in 2004-2007, which is correlated to the food catering services 7 . In Iran, the prevalence of raw vegetable parasite contaminations was about 1.94% to 68.3% in different areas 8 .
During the last decade, Adenosine Triphosphate (ATP)-bioluminescence has been increasingly applied for monitoring the surface cleanliness 9 .
The ATP bioluminescence method is an acceptable method for evaluating microbial contamination in the food industry 10 . The ATP reacts with the luciferin-luciferase enzymatic complex and the produced light is expressed in Relative Light Units (RLU) 11,12 . The ATP is a rapid, user-friendly method of quantifying the surface cleanliness within Hazard Analysis Critical Control Point (HACCP) systems 9,13,14 . The aim of this study was to investigate the measure of microbial contamination in saladcontact surfaces such as preparing tables and serving dishes by ATP bioluminescence method in restaurants of Mashhad city as the second metropolis of Iran.
The target restaurants were covered by Health Center No. 5 of Mashhad. The restaurants' hygienic and sanitary conditions were checked by the person who took the samples. The geographical location of the study area is displayed in Figure 1. In each restaurant, two samples (one from salad table and one from salad dish) were collected by swabbing an area of 10 cm × 10 cm using the Ultra Snap Swabs (Hygiena International, Watford, UK) in a day. According to the manufacturer's instructions about using the ATP bioluminescence device, the swabs were placed in the swab tube. Then, the swab tube was broken at the snap valve by bending the bulb forward and backward using the thumb and forefinger. The bulb was squeezed twice and all liquid down the swab shaft was expelled. The swab bud was bathed in liquid by softly shaking for 5-10 seconds. The swab tube was inserted into a portable luminometer "System SURE II" (Hygiena International, Watford, UK) and after closing the lid, results were read. The default setting was employed in the Hygiena luminometer when enzymes in combination with ATP molecules emitted light to the sample. This was measured by the device and its value indicated the degree of contamination or the number of microbes present in the sample. Accordingly, scores less than 10 units showed that the surface was clean (pass), in 11-29 units warned that the surface was not adequately clean (caution), and scores greater than 30 units showed that the surface was dirty (fail).

Statistical analysis
To investigate normality of the data, we used the Kolmogorov-Smirnov test. Considering that the data were abnormal, we restrained the Kruskal-Wallis H test to compare different materials and ATP values. Mann-Whitney U test was also run for comparing ATP values and two groups of dishes and tables. It is worth noting that ATP values were qualitative.

Ethical issues
This study was approved by the Medical Ethics Committee of Mashhad University of Medical Sciences No: IR.MUMS.REC.1396.241

Results
In this study, tables and dishes with different materials were studied. Figures 2 and 3 represent cleanliness status of the salad-serving dishes and salad-preparing tables made from different materials, respectively. According to the ATP bioluminescence device, 43.6% of the assessed dishes (17 samples) were clean, 38.5 % (15 samples) were dirty, 17.9 % (7 samples) were not adequately clean, 23.1 % of the tested tables passed the test (9 samples), 15.4 % were classified in the caution status (6 samples), and 61.5% failed the test (24 samples).  In this study, the highest median value of ATP was obtained for polyethylene dishes, while the lowest median value of ATP was detected in polystyrene dishes. The ATP bioluminescence results of different materials of dishes are summarized in Table 1.   No significant differences (p = 0.62) were seen between ATP bioluminescence values and materials of the salad-preparing tables.
According to the results of ATP, 53.8% of the hygienic restaurants successfully passed the cleaning test, 15.4% were specified at the caution status, and 30.8% were dirty and failed the test. However, 23.1% of the restaurants successfully passed the cleaning test, 17.3% were specified at the caution status, and 59.6% failed the test.

Discussion
The objective of this study was to confirm that application of the quantifiable methods was critical to demonstrate the quality cleanliness levels. The microbiological methods allow identifying the organism and its sensitively, but they take a long time, depend on the organism's viability and suitable transportation, and their costs are high. The microbiological method's limitation limits its daily application 15 . A real time assessment of the total surface cleanliness has been prepared in contrast to the ATP bioluminescence method, including the presence of organic debris and microbial contamination. The capability to provide results into minutes, as contrary to days for microbiological method, enables the ATP bioluminescence to be applied as a monitoring method within HACCP 9 .
In this study, sampling the salad-preparing tables and the salad-serving dishes were done before the lunchtime. In other words, they were sampled from the dishes and tables after cleaning, but our results showed that only 43.6% of the dishes and 23.1% of the tables of different materials passed the test successfully. Zambrano et al. showed that 25.37% of the surfaces cleaned by internal personnel and 68.8% of the surfaces cleaned by external personnel were considered clean. These researchers recommended application of the educational and monitoring interventions to improve the quality of cleaning 15 . Lehto et al. studied the fresh-cut vegetable production plants. The ATP bioluminescence on the surface of chopping board, packing board, steel surfaces, and inner surface of plastic box were 1595, 1578, 578, and 793 RLU, respectively. The results of this study and the present study displayed that ATP bioluminescence method was appropriate to test the surface contamination after cleaning. In the case that the samples were taken before cleaning, microbes would have overgrown on the surfaces and the ATP bioluminescence values would also have overpassed the results scale 16 . Furthermore, according to Amodio et al., surfaces exceeding the percent of chosen cut-off limit indicated a significant change in precleaning from 21.2% up to 93.1% and showed a huge rose from 5.3% to 96.5% in the postcleaning stage 17 .
According to our results, ATP bioluminescence detected that the lowest values of contamination were related to the polystyrene in dishes. Rigid PS and PS-related plastics used as food packaging materials have some of the physical characteristics of polystyrene, for instance, they have low impact strength and chemical resistance and are used to develop other food grade plastic 18 . Application of the packaging product is of great importance because they have been properly prepared, washed, and disinfected. Furthermore, the units which are carrying out the packaging process are required to comply with the approved health standards to reach the production permission; so, the contamination load on these products is low.
The results displayed that the tables made of wood and steel passed the test more successfully than the tables made of polyethylene. Stainless steel has often been selected as the most suitable surface material because it has weak adhesion characteristics and can be cleaned quickly. However, the study by Wilks et al. showed that E.coli O 157 could stay alive for long term when dried onto the surface indicating that the contamination potential factor could occur if a surface was not adequately cleaned 19 . Ak et al. conducted that "wood generally yielded fewer bacteria than plastic after cleaning and the bacteria might be recovered from wood after 12 h at room temperature in high humidity, but numbers were reduced by at least 98% and often more than 99.9%" 20 . However, the other study by Gough et al. found that treatment boards with a proprietary disinfectant was significantly more effective on plastic compared to wooden boards 21 . To compare between 3 surfaces used in our study, the study of Wanyenya et al. was applied mentioning that Campylobacter jejuni cells were able to survive longer on wooden and plastic cutting boards than the metal surfaces. This study indicated that wooden and plastic boards could retain Campylobacter spp. for a relatively long time 22 . Therefore, the food handlers should be trained over time in this regard to obtain the required knowledge 7 .
In the above-mentioned study, surfaces of a raw milk transportation tank, raw milk cooling storage tank, an equalization pasteurizing tank, a pasteurized milk storage tank, a pasteurized milk packaging tank, and a milk centrifuge were evaluated by ATP-bioluminescence method and hygiene monitoring procedures. The results showed that 100% of the studied surfaces were under inadequate hygiene conditions, while the plate count method detected only 50% of contamination based on the American Public Health Association's (APHA) recommendation. Therefore, The ATP bioluminescence method is not a direct replacement for microbiological method, but provides supplementary information and should be used as part of a confederate strategy 12 .

Recommendation
As a result of this study, some practical recommendations can be made as following:  Washing and cleaning practices should be more effective and efficient and suitable detergents should be used for different kinds of surfaces.
 Paying attention to the cleaning equipment in contact with salad-preparing surfaces and disinfecting them properly.
 Training the staffs is very important and attention to hygienic aspects must become a requirement. Hygienic condition, such as cleanness status of surfaces, must be confirmed before and after the working time.
 Staffs must be trained to use gloves during the working time and not leave the boxes and baskets on the tables.
 Surfaces should be monitored regularly and the obtained results should be recorded on a table, so that when the cleaning practices are not performed correctly the staffs would be fined for the wrongdoing.

Conclusion
The ATP bioluminescence method can be used in a few minutes to provide a real time estimate. In addition, this method can not only detect the microorganisms, but also the food residues, which is more beneficial. This is because residues of foods or organic matters indicate the effectiveness of cleaning and hygienic procedures. Furthermore, presence of microorganisms has potential impact on the public health. However, this method is not a reliable replacement for measurement of microbial contamination.