Could food or food contact surfaces be the favourable hideouts for Listeria monocytogenes in Perak, Malaysia?

Chen, S.N., Yap, M.L., Kuan, C.H., Son, R. and Saw, S.H. Department of Allied Health Sciences, Faculty of Science, Universiti Tunku Abdul Rahman (UTAR), Jalan Universiti Bandar Barat, 31900 Kampar Perak, Malaysia Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman (UTAR), Jalan Universiti Bandar Barat, 31900 Kampar Perak, Malaysia Department of Food Science with Nutrition, Faculty of Applied Sciences, UCSI University, Cheras 56000, Kuala Lumpur, Malaysia Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

L. monocytogenes is able to adapt to extreme environments, such as surviving in a wide range of pH condition from 4.5 to 9.5; multiplying at low refrigeration temperature as -1 o C and remaining viable in high salt condition at 40% w/v (Chasseignaux et al., 2002;Bucrieser et al., 2003;Liu, 2006;Meloni et al., 2009;Al-Nabulsi et al., 2015). Due to its durable characteristics, this extremophile grows easily on various food surfaces and forms biofilm: an aggregation of micro -colonies on the surfaces which enables substrate exchange, dissemination of metabolic products and elimination of toxic end products in order to sustain the growth of the bacteria communities (Donlan, 2001). The microbial biofilms immerse in a self-produced matrix of extracellular material, which consists of a conglomeration of different types of biopolymers known as extracellular polymeric substances (EPS's) (Donlan and Costerton, 2002). These structures enable a formation of strong adhesion of Listeria spp. to the surfaces and play a role in protecting the bacteria from antimicrobials invasion and shearing forces, as well as in tolerating the components of both the innate and adaptive immune response and resists phagocytosis. Thus, biofilms production is an important source of L. monocytogenes contamination that can lead to food spoilage or transmission of disease (Lundén et al., 2000).
In Malaysia, several prevalence studies had been reported for the presence of L. monocytogenes in various foods such as meat, chicken offal, beef offal, beef patties, vegetables, salad and ready-to-eat food (Arumugaswamy et al., 1994;Tang et al., 1994;Ponniah et al., 2010;Marian et al., 2012;Wong et al., 2012;Kuan, Goh, Loo et al., 2013;Kuan, Wong, Pui et al., 2013;Marian et al., 2019). Contamination of the food could occur at all stages in the food chain especially at the surfaces of the food processing plant and during the distribution of the end products at the premises. This happened in the year 2015, when our Ministry of Agriculture and Food Industry seized the import of L. monocytogenes contaminated apples from Bidart Bros in Bakersfield, California (Malay Mail, 2015). The cause of contamination might be due to the occurrence of crosscontamination of the food in the farm with food contact surfaces of food processing plants or retail premises. Thus, this study aimed to investigate the occurrence of L. monocytogenes in food and on food contact surfaces in Perak, Malaysia by the culture-method and duplex-PCR (d-PCR) molecular method.

Sample collection
From August 2018 to August 2019, a total number of 322 samples (170 food samples and 152 food contact surface samples) were randomly collected from six food premises, two food processing plants, four hypermarkets, a wet market and a night market in Perak, Malaysia. The types of samples collected are summarised in Table 1.

Food samples
Food samples were purchased freshly from the hypermarkets, wet market and night market. Food from different categories was picked randomly from different stalls of each sampling site. The raw, minimally processed and processed foods were kept separately in cold storage boxes whereas ready-to-eat foods were kept under room temperature during the transportation back to the laboratory for analysis.

Source/Total
Samples Collected Food (170) Raw Food (38) Vegetables (23), Fresh meat and seafood (10), Bean sprouts (5) Minimally Processed Food (33) Minced meats (2), Pre-cut meats (5), Pre-cut fishes (6), Pre-cut vegetables (18), Bean curds (2) Processed Food (6) Quick-frozen meat products (4) Vacuum-packed meat products (2) Ready-to-eat (RTE) Food (93) Sandwiches (13) Swab samples from food contact surfaces were collected according to the protocol described by Public Health England (2017) with slight modifications. Individually packed sterilised cotton swab was premoistened with sterilised 0.85% (w/v) of saline solution (Merck, Germany). Then, a sterilised swab template of size 10 × 10 cm was placed on the tested area and was swabbed from left to right, up to down and at diagonal sides for 30 s. Rotation of the swab was performed during the collection process. The swabbed cotton was placed into a labelled tube containing 10 mL of 0.1% (w/ v) peptone water (LAB M, United Kingdom). Collected samples were stored in a cold storage box during transportation to the laboratory. The samples isolation was then performed within 24 hrs.

Pre-enrichment, enrichment and purification steps
The detection and isolation of L. monocytogenes from the collected food samples was performed based on the procedure described by Kuan, Goh, Loo et al. (2013) and Kuan, Wong, Pui et al. (2013) with slight modifications. In brief, 25 g of food sample (from section 2.1.1) was placed in a sterile stomacher bag and homogenised with 225 mL of Listeria Enrichment Broth (LEB) (Merck, Germany) for 2 min using stomacher machine of BagMixer® 400P (Interscience, France). The suspension of 250 mL was then incubated for 4 hrs at 30 o C, before adding the selective supplements agents: acriflavin, 10 mg/L, sodium nalidixate, 40 mg/L, cycloheximide 50 mg/L (Merck, Germany). Incubation was performed for 44 hrs at 30 o C.
On the other hand, the collected swab sample of food contact surfaces (from section 2.1.2) that had immersed in 0.1% (w/v) peptone water was vortex for 3 min. A 1 mL portion of the suspension was then transferred and homogenised in 9 mL of LEB and incubated for 4 hrs at 30 o C before the enrichment supplements were added. The samples were further incubated for 44 hrs at 30 o C.
After 48 hrs of incubation, 0.1 mL of broth for all samples were spread plated on PALCAM agar (Oxoid, UK) and incubated for another 48 hrs at 30 o C. Five presumptive colonies were picked from each PALCAM agar plate and sub-cultured onto Tryptic Soy Agar (TSA) (Merck, Germany). TSA agar was then incubated for 48 hrs at 30 o C. This step was performed to purify the Listeria colonies before DNA extraction was performed.

Extraction of DNA
The boiled cell method was used to extract the DNA of the presumptive colonies from TSA plates, as described by Kuan et al. (2017). Briefly, one full loop of culture was scrapped from the TSA plate and resuspended in 200 μL of sterile distilled water. The suspension was then vortex prior to the boiling step at 100 o C for 10 mins. The cell was then cooled at -20 o C for 10 min before it was centrifuged at 13,400 x g for 3 min. The supernatant was subjected to PCR to identify the Listeria spp. and L. monocytogenes strains.

Duplex Polymerase Chain Reaction (d-PCR)
A total of 1,842 presumptive colonies were isolated and verified using duplex-polymerase chain reaction (d-PCR). d-PCR was carried out using two primer pairs: LI1 and U1, sequences as LI1-5' CTC CAT AAA CGT GAT CCT 3' and U1-5' CAG CMG CCG CGG CGG TAA TWC 3'; as well as LM1 and LM2, sequences as LM1-5' CCT AAG ACG CCA ATC GAA 3' and LM2-5' AAG CGC TTG CAA CTG CTC 3'. The first pair was a genus-specific primer for Listeria spp. which amplified at 16S rRNA gene with the size of 938 bp, whereas, the second pair was a species-specific primer for L. monocytogenes amplified at hlyA gene with the size of 702 bp. Both primer pairs were synthesised by Apical Scientific Sdn. Bhd. d-PCR amplification was performed as described by Kuan, Goh, Loo et al. 2013 andKuan, Wong, Pui et al. (2013) with slight modifications. d-PCR was conducted in a reaction mixture of 25 µL which contained 5 µL of 5X PCR buffer, 1.5 µL of 25 mM MgCl 2 , 0.2 µL of 10 mM deoxynucleoside triphosphate mix, 0.3 µL of 1.5U Taq DNA Polymerase, 0.5 µL of LI1 primer, 0.5 µL of U1 primer, 0.5 µL of LM1, 0.5 µL of LM2, 14.0 µL of sterile distilled water and 2.0 µL of DNA template (supernatant from the extraction of DNA). All reagents used in the PCR amplification were obtained from Promega (Research Instruments, USA). L. monocytogenes ATCC 19115 was used as a positive control for each PCR assay. The PCR conditions used was as such: initial denaturation at 94 o C for 5 mins, followed by 30 cycles of denaturation at 94 o C for 30 s, annealing at 53 o C for 1 min and extension at 72 o C for 2 mins, followed by a final extension step at 72 o C for 7 mins. The thermal cycling reactions were performed using Thermal Cycler (Matrioux, Malaysia). Then, the PCR products were subjected to 1.5% agarose gel electrophoresis in 0.5X of Tris-Borate-EDTA (TBE) buffer at 100V for 45 min. The gel was then stained with 3X gel red and visualised under a gel imager (Bio-rad, USA). A 100 bp DNA ladder (Vivantis Technologies, Malaysia) was used as a DNA marker to estimate the size of amplified PCR products.

Detection of Listeria monocytogenes in food and on food contact surfaces
The presence of L. monocytogenes was detected on PALCAM agar plate which exhibited colonies of greygreen colour with a black centre (Figure 1). The genus of Listeria was confirmed via PCR amplification of 16S rRNA which yielded 938 bp in size, while the presence of the species was confirmed via amplification of its haemolysin gene that encodes for listeriolysin O (hlyA) gene at 702 bp product   (Figure 2).

Prevalence of Listeria monocytogenes in food and on food contact surfaces
Out of 170 food samples collected, 38 (22.35%) were raw food, 33 (19.41%) were minimally processed food, 6 (3.53%) were processed food and 93 (54.71%) were ready-to-eat food. Among 152 swab samples of contact surfaces collected, 93 (61.18%) were from direct food contact surfaces and 59 (38.82%) were from indirect food contact surfaces (Table 1). The prevalence of Listeria, as well as L. monocytogenes in the food samples and contact surfaces, were tabulated in Table 2. Listeria was found in 69 out of the 322 total samples (21.42%) collected and more than half of these samples were positive for L. monocytogenes (n=41/69, 59.42%).
Among the 26 L. monocytogenes positive food samples, processed food showed the highest prevalence at 33.33% (2 out 6 samples), followed by minimally processed food which accounted for 31.25% (10 out of 33 samples), raw food at 26.32% (10 out of 38 samples) and ready-to-eat (RTE) food at 4.26% (4 out of 93 samples). Nevertheless, Listeria was found in about 18.28% (17 out of 93 samples) in RTE food, which accounts for some attention on its presence. On the other hand, direct food contact surface was found to be more easily contaminated by L. monocytogenes with a prevalence of 11.83% (11 out of 93 samples) compared to indirect food contact surfaces, where these bacteria were detected at a prevalence of 6.78% (4 out of 59 samples).

Discussion
Listeriosis is a life-threatening foodborne disease that is caused by the ingestion of food contaminated by L. monocytogenes. The questions ponder about the source of the contamination. It is postulated that the bacteria have come in contact at the food processing plant and start multiplying during the storage and at the retail level. In this study, it has been proved that both sources of food and food contact surfaces are at risk of getting exposed to Listeria contamination. Processed food, such as the frozen chicken slices and vacuumpacked smoked duck meat was observed to have the highest prevalence of L. monocytogenes, compared to minimally processed food, raw food and RTE food. On the other hand, direct food contact surfaces recorded a higher contamination level of L. monocytogenes than indirect food contact surfaces.
Processed food has a higher probability of being contaminated due to its long processing procedures needed. Before turning fresh food into food products, one or a combination of various processes, including washing, chopping, pasteurising, freezing, fermenting,   , 2008). Despite the harsh treatment process involved, L. monocytogenes has the ability to strive for survival. Since most of the processed food is stored at low temperature in order to maintain its freshness, it may in turn create a favourable growth condition for the bacteria to proliferate. This observation was supported by a study conducted by Wong et al. (2012) who had reported that 22.33% of burger patties were detected positive for L. monocytogenes. In Assiut city of Egypt, El-Malek and colleagues (2010) had found a lower prevalence of L. monocytogenes detected in frozen meat and chicken samples. In addition, Marian et al. (2012) had reported that 33.3% of the burger samples collected from local wet markets, mini markets and supermarkets in Selangor, Malaysia were contaminated with L. monocytogenes. Thus, frozen processed meat products are a favourite reservoir for the growth of the foodborne pathogen.
Minimally processed food is another food material that has found to be easily contaminated by L. monocytogenes. According to the food classification tool (NOVA classification) developed by Monteiro et al. (2016), minimally processed food, including poultry, meat, seafood steaks, fillets, fresh-cut fruits and vegetables, as well as fresh or dried herbs such as mint or thyme, is a natural food product that undergoes mild processed without the addition of flavouring, salt and sugar. The mild processing steps involve sorting, washing, peeling, slicing, cutting, grinding and removing inedible parts of the fresh produce (Bansal et al., 2015). Again, the surfaces of grinders and some machinery in the process could be the potential sources of Listeria contamination due to the ideal temperature of the production area and improper cleaning of the complicated structure of machinery. In this study, L. monocytogenes was found in pre-cut vegetables (5/18), pre-cut meats (2/5), pre-cut fishes (2/6) and bean curds (1/2). The results were comparable with the study conducted in Japan and Turkey. Researchers from these two places had found the presence of L. monocytogenes in minced beef (12.2%) and ground beef (7.2%), as well as chicken meat samples (17.8%) from the retail premises (Inoue et al., 2000;Kalender, 2011).
On the other hand, raw food, either animal or plant sources, is natural or unprocessed foods that are obtained directly from farms (Poti et al., 2015;Monteiro et al., 2016). Although the percentage of Listeria spp. in the samples was less than 30%, its presence in 10 out of the 38 samples was a concern of listeriosis threat. Raw food might get contaminated from the soil, wastewater and faeces. Soil treated with artificial fertilizers creates a suitable growth environment for Listeria compared to soil treated with natural fertilizers (Szymczack et al., 2014). On the other hand, improper handling of wastewater management at the farms is often the case for the occurrence of cross-contamination (Lyautey et al., 2007). Thus, proper washing and cooking, preferably at 74 o C are encouraged to reduce the risk of contracting listeriosis.
In this study, 4.26% of the RTE food samples derived from fruits (1/12), sandwiches (1/13), desserts (1/25) and cooked food (1/27) were detected with L. monocytogenes. RTE vegetables, fruits and sandwiches are common sources for the sprouting of foodborne pathogens due to their zero heating processes and openshelf storage in low refrigeration temperatures. Ponniah and colleagues (2010) had reported that 22.5% of RTE vegetables were positive for L. monocytogenes. Also, studies by Jamali et al. (2013), Leong et al. (2014) and Mureddu et al. (2014) had reported the presence of L. monocytogenes in RTE food samples at between 5.0 to 30.0%.
Elimination of Listeria poses a great challenge as it has the ability to strive for survival in harsh conditions. Hence, the strategy to mitigate the growth of the pathogen is to first detect the presence of L. monocytogenes in food processing plants and food contact surfaces before it reaches the final products. In this study, Listeria was found on the surfaces of the food processing machines such as food processing machines, conveyor belts, working benches for food processing and preparation, as well as on the surfaces of cutting boards. The highest prevalence of L. monocytogenes was found in food processing machines (6/19) such as roller bar, orientation and cooling machine. This observation was supported by Lundén et al. (2003), who stated that the complex machines in the processing lines were at high risk to be contaminated by L. monocytogenes due to irregular sanitisation of the food processing surfaces. Also, a low incidence of L. monocytogenes was found on the surfaces of processing tools used to produce sausages (Chevallier et al., 2006;Gounadaki et al., 2008). Following these incidences, the researchers in the US had revealed the presence of L. monocytogenes in the refrigerated food processing surfaces and equipment despite the regular cleaning and disinfecting (Carpentier and Cerf 2011;Hoelzer et al., 2011). These studies had once again proved the high adaptability of L. monocytogenes in harsh environment.
Besides the direct food contact surfaces, non-direct food contact surfaces are a favourable choice for the growth of L. monocytogenes. In the food production area, floor and drains are retained in a cold and wet atmosphere, which create a notably favourable habitat eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources FULL PAPER for L. monocytogenes to reside. This is seen in this study, whereby Listeria was detected in an area of display racks and cleaning cloths. This was also seen in the study by Leong et al. (2014), who had found a prevalence of 4.4% of environmental samples collected from 48 food business operators in the Republic of Ireland were positive for L. monocytogenes. On the other hand, contamination rate of L. monocytogenes on non-food contact surfaces such as floors, walls, drying rooms and steaming rooms in meat processing line were astonishingly high in the range between 11.0% to 25.0% (Thévenot et al., 2005;Mureddu et al., 2014).
In this study, the overall prevalence of L. monocytogenes detected in foods and on food contact surfaces from the food processing plant and food premises were low. Nevertheless, there is still a need for active surveillance in order to monitor the real scenario and to create awareness on the importance of cleanliness status in food and food contact surfaces from food processing plants and premises.

Conclusion
In conclusion, the overall prevalence of L. monocytogenes in food and food contact surfaces from food processing and food services environment are lower in Perak compared to studies in Selangor, Malaysia. However, this might pose a risk of listeriosis outbreak if further action is not taken as it will act as the route of transmission to the consumers. Food handlers, especially in the food industry and food premises have to perform robust surveillance, risk assessment and practice effective sanitary procedures in order to reduce the risk of growth of L. monocytogenes. It is important that efforts continue to understand the ability of the organism to survive and multiply under adverse conditions, and this knowledge can be used to design new control strategies.