Preliminary screening and microbiological evaluation on the environmental hygiene for galley equipment, safety equipment and cabin common facilities of a local airline in Malaysia

Preliminary screening and microbiological evaluation on the environmental hygiene for galley equipment, safety equipment and cabin common facilities of a local airline in Malaysia Wing, G.K., Rollon, D.W., Kuan, C.H., Ajau, D., Nor-Khaizura, M.A.R., Hasan, H. and Son, R. Department of Food Science, Faculty of Food Safety and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia Department of Food Science with Nutrition, Faculty of Applied Sciences, UCSI University, Cheras 56000, Kuala Lumpur, Malaysia Faculty of Health Science, Universiti Teknologi MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia


Introduction
The odds of having difficulty conducting research in the confinement of commercial aeroplanes that fly 35,000-40,000 feet above sea level is undeniably high. Such difficulty may be contributed and limited by stringent regulatory requirements that govern the security and safety of the aviation industry. Therefore, as industrial skilled players, this study presented a rare opportunity which was aimed to conduct preliminary screenings that determine the microbial quality of galley equipment, safety equipment and common facilities of aeroplanes operated by a local airline in Malaysia.
The aerobic mesophilic plate count is a conventional method of microbial analysis to estimate the presence of cells based on their ability to give rise to colonies under specific conditions of nutrient medium, temperature and time. It is also intended to indicate the level of microorganisms in a product. It is considered the most eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER widely used technique or tool for evaluating microorganisms in foods (Brackett, 2014). Whichever method is selected or used to achieve the primary purpose of analysis may be subjected to accuracy, reproducibility, reliability, specificity, and sensitivity (Tunung et al., 2012). Therefore, it is also necessary to conduct aerobic plate count as it indicates the level of microorganisms in a product (Maturin and Peeler, 1998). According to Saarela (2007), the plate count technique is based on the reproduction of bacterial cells on agar plates. The plate count technique is the traditional method used for the quality assurance of probiotic products. Even though this method provides information about the microbial load in food samples, it is not without any limitations. The first drawback is that the standard plate count only tells how many cells are but not what kind of cells are present. Secondly only relatively rapidly growing aerobic organisms such as bacteria are enumerated (Brackett, 2014). On one hand, aerobic mesophilic plate counts (AMPC) are considered poor indicators of food safety in many cases because they do not directly correlate to the prevalence of pathogens or toxins. While on the other hand, subject to the condition of the product, AMPC can be valuable in accessing the microbial quality and organoleptic acceptability of foods (Pianetti et al., 2008). Therefore, conditions used in standard plate count may not be able to enumerate many fungi and as a result other important organisms, intended or otherwise, are missed out by this procedure.
Interestingly, various studies reported that pH (Therion et al., 1982;Cole et al., 1990;Presser, 1997;Koutsoumanis et al., 2004) and temperatures may have influenced the growth limit of pathogenic bacteria (Rocourt and Cossart, 1997;Shachar and Yaron, 2006;Gandhi and Chikindas, 2007). Kim et al. (2018) postulated that the optimal levels for bacterial growth rate were at pH 9 and 35˚C. These 2 factors will determine the growth rate and responsiveness of microorganisms. Russell (2005) was using the standard plate count method for enumerating microorganisms from food samples based on the presumption that organisms are able to multiply in cultural media containing agar to form colonies. It is also assumed that each colonyforming unit (CFU) represents a single bacterium that has grown in or on the medium in question. Given exposure to appropriate temperature and atmosphere, the mass of cells is produced and can be observed by sight so that the colonies can be counted.
The galley equipment (GE), safety equipment (SE) and common facilities (CF) are regularly utilized in the course of handling and serving food as well as replenishing amenities in the cabin common facilities onboard the aeroplane. These processes are undoubtedly exposed to a high potential of direct contamination and other cross-contamination factors. Such adulterated conditions serve as possible food contamination that leads to food poisoning.
The results of this study should be able to capture the awareness of the importance of microbial quality evaluation because it is closely associated with food handling hygiene and food handling practices among flight attendants. To the best of our knowledge, this is the first study of its kind and of which the outcome can be used as a baseline reference for future research development. Therefore, this study was a novel attempt to fill the missing gap of knowledge on such a setting in the Malaysian aviation industry.

Samples collection
A total of 112 swab samples (n = 112) were collected from five selected inbound flight sectors in accordance with the aircraft types and flight time range. Table 1 entailed details of swab sample distribution. Upon arrival at Kuala Lumpur International Airport, the targeted swab samples were immediately transported to the Food Safety and Quality Laboratory at Food Science and Technology faculty, UPM Serdang. Each cotton swab was placed individually in a small sterile zip-type transparent polyethylene plastic bag, 30×60 mm. All of the individually packed swab samples were kept cool in another large sterile double zipper transparent plastic bag, 144×160 mm. The large sterile plastic bag was placed in a mini cooler bag and transported to the laboratory within 4 hrs upon arrival of aeroplanes at Kuala Lumpur International Airport.

Microbiological analysis
Mesophilic aerobic bacterium; Escherichia coli, Vibrio, Salmonella and coliforms were enumerated using the conventional method (Morton, 2001). Buffered peptone water (BPW) (Oxoid, UK), measured and poured into 10 mL universal bottles as per quantity required and was autoclaved at 121˚C for 15 mins. The cotton swab samples were cut at both ends and placed into the prepared sampling bottles. Every sample was briskly shaken to harmonize the diluent before being incubated at 37˚C for 24 hrs. The incubated swab samples were then streaked onto respective agar plates and incubated at 37˚C for another 24 hrs. The agar plates were inspected for any microorganism growth. Plates that showed any form of prevalence growth were noted as positive samples while agar plates that did not show

Flight sectors selection
Five inbound flight sectors into Kuala Lumpur International Airport, KLIA were selected. One of which was an inbound long haul sector from London Heathrow International Airport. This flight sector was operated by Airbus A350-900. Two medium-haul flight sectors; an inbound sector from Mumbai (India) and another inbound sector from Narita, Tokyo (Japan) were also selected. The flight from Mumbai was operated by Airbus A330-200 and the sector from Narita was operated by A380-800. Similarly, 2 short-haul flight sectors; one international flight originated from the Surabaya sector which was flown using Boeing B737-800 and one domestic flight sector which was flown by A330-200 from Kota Kinabalu, were inclusively selected for these preliminary screening microbial analyses. Table  2 illustrates the flight sector selections.

Microbial preliminary screening result for the selected inbound flight sectors.
After the incubation period of 48 hrs, the aerobic mesophilic agar plates were inspected for microbial growth of the selective microorganism indicators. On 16 th July 2018, an Airbus A330-300 was used to fly the passengers across the South China Sea from Kota Kinabalu (BKI) into Kuala Lumpur (KUL). Such a flight sector was considered a rushing flight sector because the flight time was scheduled at 2 hrs 15 m. Taking into account that this flight sector was a short-haul flight, time was the indefinite constraint, therefore, only the common facilities were sampled for microbial analysis. According to Table 3, the preliminary screening results reported that 8 samples were positive and 6 samples were negative.
In addition, 21 samples were collected from a medium-haul flight sector that originated from Mumbai (BOM), India on 28 th July 2018 which travelled using A330-300. The swab samples were taken from 6 galley equipment, 7 safety equipment and 8 cabin common facilities. As indicated in  Narita Tokyo (NRT) was another inbound mediumhaul flight that departed from Japan. This flight was operated by the superjumbo Airbus A380-800 on 10 th October 2018. A total of 30 swab samples were taken from galley equipment only. The preliminary screening schedule in Table 3 showed 24 samples were positive. Only 6 samples were reported as negative. The 5 th microbial preliminary screening analysis in which another 30 samples were sampled from Surabaya (SUB), Indonesia to Kuala Lumpur (KUL), was a short-haul inbound flight sector. Twenty swab samples were taken from galley equipment, 8 safety equipment and 2 cabin common facilities. The aircraft designated for this flight was a narrow-bodied aeroplane with a single aisle, Boeing B737-800. Table 3 showed only one sample was reported negative while 29 samples were observed as positive. The positive samples of the galley equipment, safety equipment and common facilities were reported at 19, 8 and 2 samples, respectively.

Discussion
An agar plate was considered as a positive sample if visible microorganism growth was detected. When no visible microbial growth was detected, then the plate count was counted as a negative sample. Table 3 illustrates that the short flight sector from Kota Kinabalu (BKI) showed 8 positive and 6 negative samples. The 21 and 17 samples taken from a medium-haul flight sector Mumbai (BOM) and a long haul flight London (LHR) were denoted as all positive. The 24 samples derived from another medium flight sector from Narita, Tokyo were positive. Lastly, the results from another short-haul inbound flight sector from Surabaya produced 29 positive samples. Therefore, it was concluded that the preliminary microbial screening results showed that 99 (88.39%) of the swab samples were denoted as positive with at least one microbial indicator and 13 (11.61%) were negative swab samples free from all of the microbial indicators.
From the results of the plate counts, it was quite an alarming finding that the microbiological quality of the equipment and common facilities were highly contaminated as reported by the prevalence of microorganism indicators.  This did not rule out the potential food poisoning incident caused by spoilage in food and water (McMullan et al., 2001). Consumption of contaminated food by pathogenic bacteria like Salmonella Enteriditis (SE) can be deadly. A study case by Ogata et al. (2009) found that a passenger died due to consumption of a small piece of food contaminated by Salmonella Enteriditis (SE). The classic example demonstrating the devastating consequences of an in-flight food poisoning outbreak was recorded on 3rd February 1975. According to Eisenberg et al. (1975), 1 crew member and 344 passengers contracted a gastrointestinal illness which brought 142 of the passengers to the hospital. The investigation reported that Staphylococcus aureus and food handlers were the main agents of food contamination.
Coliforms in general indicate the presence of faecal contamination. The primary sources of coliforms are from the animal intestinal tract and the other is from soil vegetation and insects. Unlike most microbiological research that took samples from the commonly regulated environment like school canteens, college cafeterias, restaurants etcetera, the samples for these particular inflight food safety microbiological analyses were taken from the confinement of aeroplanes that were travelling at 35,000-40,000 feet above sea level. Numerous research have been conducted to assess the microbiological quality of surfaces (Flores et al., 2011;Rodríguez et al., 2011;Garayoa et al., 2016;Tóth et al., 2018;Touimi, et al., 2019;Sibanyoni et al., 2019;Osaili et al., 2020). There were also many studies that quantify the microbiological quality of ready-to-eat foods. While microbial assessment on the surfaces can be performed to determine the effectiveness of cleaning and sanitization, the microbial analysis of ready-to-eat foods can be conducted to determine whether or not the samples used for the testing are contaminated by any selected microorganism indicators (Khater et al., 2013;Syne et al., 2013;Nahar and Mahyudin, 2018;Petruzzelli et al., 2018;Mengistu and Tolera, 2020).
A total 112 surface swab samples have been selectively taken from five different inbound flight sectors. This hands-on study was conducted in reference to a previous study by Crosby et al. (2008) in which they assessed the microbiological quality of three food contact surfaces and one non-food contact surface at childcare centres. They chose E. coli and coliforms as the bacterium indicators for their microbial assessment. However, in addition, and concurring with their selected bacterium indicators, media for Vibrio and Salmonella were included as additional microorganism indicators for this microbiological preliminary screening. Even though the results of a preliminary screening were as simple as to categorically notate them as positive or negative samples, this approach was sufficient to determine and describe whether the sampling areas were scientifically contaminated or not. In reference to Table 3 and Table 4, it was reported that the total positive samples from all the five inbound flight sectors selected for the swab sampling were 99 (88.37%). Table 4 showed coliforms were detected as the most prevalent bacteria, 87 or 34.39%. It was found that positive samples from the galley equipment, safety equipment and cabin facility were 64 (57.14%), 20 (17.86%) and 28 (25%), respectively.
The prevalence of E. coli in the AMPC suggested faecal contamination from the hands of the flight attendants onto the galley and safety equipment. Flight attendants are known for their multi-tasking ability. When flight attendants clean and replenish the lavatory amenities, it is a common practice for them to open the storage compartment in the lavatory where the amenities are located. By doing so, cross-contamination is made possible because bacteria can live on surfaces, and they are not seen by their naked eyes. This condition appears to be in agreement with findings by Edema and Omemu where they reported that prevalence represents a lack of cleanliness in food handling as well as improper food storage (Edema and Omemu, 2004). The prevalence of microbes is also an indication of possible contamination from passengers' hands because they have open access over the cabin common facilities. As illustrated in Table  5, although the prevalence of E. coli and Vibrio were at 56 (12.5%) and 49 (10.94%), the coliform contamination proved to have been present at a high 87 (19.42%) of the total plate counts. Concurrently, Salmonella was present in 43 (9.6%) of the total plate counts.
Vibrio spp. is mostly marine in origin and its taxonomy is continuously being revised due to the addition of new species (Etinosa et al., 2008), its infection has also been commonly found as foodborne infections in countries within Asia (Sutherland and Varnam, 2000). These bacteria can also be found on  (Chai et al., 2008;Yang et al., 2008;Tunung et al., 2010). In agreement with the study conducted by Wilks et al. (2005), such bacteria as E. coli can survive on the surface of steel for a period as long as 28 days in refrigerated and non-refrigerated temperatures. The surfaces of the swab sampling areas taken for this study were no exceptions.

Conclusion
Considering the sensitivity of the working environment in the aviation industry, the opportunity to conduct this preliminary microbial analysis was indeed a privilege that was hardly taken to task. This was truly a rare opportunity that contributed to the body of knowledge that befitted the aviation industry. This research fundamentally focused on elementary microbial evaluation. Therefore, the outcome of this microbiological analysis is set as a milestone that may be further improved by other researchers within and outside the industry. The parameters as prescribed in the HACCP management system should be constantly reviewed to include consistent monitoring enforcement to ensure that the critical control points of food safety are not abused. Personal hygiene, which includes correct washing of hands and proper sanitizing of galley equipment is imperative to prevent contamination and cross-contamination. Flight attendants who regularly replenish the lavatories should be more concerned about the sterility of lavatories. To diligently sanitize the touching points and areas such as lavatories, doorknobs, latches, switch buttons, facial mirrors and water faucets should become their habitual practices each time they utilize and replenish the lavatories. The same attitude is required of them when managing other areas of the galley and safety equipment. Therefore, highlighting and creating awareness of the importance of practising good housekeeping, maintaining meticulous personal hygiene practices among the flight attendants are imperative in the course of performing their in-flight duty. Failing to do so may pose a high risk of cross-contamination that potentially lead to food poisoning. Any forms and elements that contribute to food poisoning are health risk parameters that society cannot afford to bear.

Conflict of interest
This study was consented to by the department manager. The due process of collecting swab samples did not in any way interfere with the core duties of the flight attendants. There was no conflict of interest.