IJERD – International Journal of Environmental and Rural Development (2020) 11-1

Research article
erd

Evaluation of Bacterial Contamination Levels in Pickles
Sold at Wet Market in Cambodia -Part 2- Detection ofSeveral Food-poisoning Bacteria of 48 Samples
from Phnom Penh
SHUKI MURAMATSU*
Faculty of Life and Environmental Sciences, Showa Women’s University, Tokyo, Japan
Email: s-muramatsu@swu.ac.jp

MASATAKA UCHINO
Faculty of Life Sciences, Tokyo University of Agriculture, Tokyo, JapanSOKLY SORM
Graduate School of Agriculture, Tokyo University of Agriculture, Tokyo, JapanYURI TANIKOKA
Faculty of International Agriculture and Food Studies, Tokyo University of Agriculture, Tokyo,JapanDAIKI OKA
Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo, JapanYOSHIKI MURAMATSU
Faculty of Regional Environment Science, Tokyo University of Agriculture, JapanTORU NAKAJIMA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Japan

MOTOE SEKIDO
Department of Food and Nutrition, Yamanashi Gakuin Junior College, Tokyo, Japan

TAKAHIKO NAKAMURA
Faculty of Regional Environment Science, Tokyo University of Agriculture, JapanCHIM CHAY
Faculty of Agro-Industry, Royal University of Agriculture, Phnom Penh, Cambodia

MACHITO MIHARA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Japan
Received 15 January 2020

Accepted 23 April 2020(*Corresponding Author)

Abstract Some pickles transported from the local province are sold in the wet markets ofPhnom Penh, Cambodia. Therefore, products sold in Phnom Penh can be considered
representative samples that reflect the approximate levels of bacterial contamination in thefood supply chain of Cambodia. In this study, we conducted a microbiological inspection of
the food samples from Phnom Penh to investigate the seriousness of the unsanitary conditionsof food products. Eight kinds of pickles were purchased from three wholesale markets oneweek apart on two separate occasions. A total of 48 samples were used for microbial testing,and the values of Brix, pH, and salt content of these samples were measured. Although all
food samples were purchased from the same markets and vendors, several pickles showed
different results in the measured values between the first and second purchases. To determine
the contamination level of the pickles, we targeted general viable bacteria, coliforms, fungi,Ⓒ ISERD

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Salmonella, Staphylococcus aureus, and Bacillus cereus. The total number ofmicroorganisms detected by the aerobic plate count showed that 15 of 48 samples contained
more than 7 log10 CFU/g of sample. Pickled onion and mustard showed relatively higher
number of microorganisms compared to other products. Of the 48 pickles, 58.3% werecoliform positive, 41.7% of the samples were Salmonella positive, and 83.3% and 37.5%
were S. aureus and B. cereus positive samples, respectively. These results indicate that theproducts from the wholesale market of Phnom Penh are significantly contaminated withbacteria. In general, food contamination result from raw materials, cross-contamination, andpoor water quality. Thus, it is important to educate food handlers regarding the practicalknowledge to ensure proper food sanitation practices. The results of this study will be usefulto reveal the cause of food contamination and to develop appropriate countermeasures.
Keywords food contamination, food poisoning bacteria, wet market, pickles

INTRODUCTION
Pickles are ready-to-eat (RTE) products and are very popular to Khmer people in Cambodia.
However, due to the absence of heat sterilization prior to consumption, the safety of the products isa major concern. Our previous study had found that several pickles sold at the wet markets ofKampong Cham and Phnom Penh (PP), Cambodia were unsanitary. In addition, the hygiene practicesof the local wet market were very poor, which subsequently lead to food contamination. We speculatethat these unsanitary conditions of food and food-processing and the selling environment werederived from the food handlers who had limited awareness and knowledge on food hygiene.
The vegetables that are often the raw material for pickled foods can harbor pathogens such as
Escherichia coli and Salmonella (Francis, 1999; Matthews, 2017; Skočková, 2013). Oliveira et al.
(2011) reported that there were Salmonella and thermotolerant coliforms in RTE vegetables. Further,
there are several kinds of food spoilage microbes and foodborne pathogens that account for themajority of foodborne diseases. The World Health Organization reported that the leading causes offoodborne deaths included Salmonella and enteropathogenic E. coli (WHO, 2015). B. cereus is awell-known food poisoning bacteria. B. cereus has caused food-borne outbreaks via a large varietyof foods in many countries (Tewari, 2015). S. aureus is a ubiquitous bacterium that causes potentiallyfatal infections (Kadariya, 2014). To assess the safety and quality of food products in Cambodia, theinformation on the actual hygiene practices for food products served to local Khmer people is very
important. However, scientific reports on food safety in regards to the pickle products in Cambodia
are nearly absent except for one report by Chrun et al. (2017). Therefore, it is necessary to accurately
assess the microbiological safety of food products in Cambodia. In order to determine the foodhygiene level in Cambodia, we investigated the major foodborne pathogens and food spoilage
microbes in pickled products. The level of microbe contamination in samples we tested reflect thesanitary conditions of the wet market. In this study, we investigated the pickle products sold in PP.
Because some products are transported from the local province to PP, the products sold in PP
represent a model of the food supply chain in Cambodia. Due to the large population of PP, microbialfood contamination leads to foodborne disease outbreaks.
OBJECTIVE
The objectives of this study were 1) to analyze the current status of food and microbiological qualityof pickles sold in PP, 2) to assess the substantial food hygiene problem in Cambodia.
METHODOLOGY
Evaluation of the Quality of Pickle ProductsⒸ ISERD

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Eight kinds of common pickle products were purchased from three major wholesale markets of PP.
The popular pickle products were selected based on interviews with Khmer people. They werecollected in March, 2019. In the following week, the same pickles were purchased from the sameshops. In total, 48 samples were collected and used in this study (Table 1). The samples were stored
at 4°C for one day. The values of Brix, salt concentration, and pH of the samples were measured.
Table 1 Pickles sample used in this study
MarketSample

Product name

SampleProduct name

A/B/CA
B
C
D

Fermented papaya (sliced)

EFGH

Pickled mustard (small)Fermented cucumber
Pickled young onion
Pickled mustard (big)Pickled cucumber
Sweet salty radish
Kong chay

Microbiological Examination of Pickle Products
Ten grams of the food sample was mixed with 90 mL of Maximum Recovery Diluent (Merck KGaA,Darmstadt, Germany) solution in a sterilized storage bag. To make a homogeneous suspension, thesample was homogenized by a stomacher. After stomaching, 1 mL of the sample suspension wasmixed with 9 mL of Maximum Recovery Diluent solution in a sterile tube. The serial dilution samples
were prepared and used for testing. For the detection of S. aureus and Salmonella, Buffered Peptone
Water (Merck KGaA, Darmstadt, Germany) solution was used for sample preparation. The standard
agar medium (AS ONE, Osaka, Japan) was used for the general viable count. Desoxycholate agar
medium (AS ONE, Osaka, Japan) was used for the detection of coliform. Potato Dextrose Agarmedium (AS ONE, Osaka, Japan) was used for fungi. CHROMagarTM Salmonella medium (KANTOCHEMICAL, Tokyo, Japan) was used for Salmonella. Mannitol salt agar medium with egg yolk
(KANTO CHEMICAL, Tokyo, Japan) was used for S. aureus. BACILLUS CEREUS AGAR BASE(KANTO CHEMICAL, Tokyo, Japan) with Oxoid Polymyxin B Supplement (KANTO CHEMICAL,Tokyo, Japan) and egg yolk was used for B. cereus. 10 μL of the dilution samples aliquoted ontoplates. For general viable counts and the detection of coliform, S. aureus, plates were incubated at
35°C for 48 hours. For the detection of Salmonella and B. cereus, plates were incubated at 35°C for
24 hours. For the detection of fungi, plates were incubated at 30 ℃ for 5 days. All experiments wereconducted three times independently.
RESULTS AND DISCUSSION
Evaluation of the Quality Variation of Pickle Products
Eight kinds of popular pickle products for local Khmer people were selected and purchased fromthree major wholesale markets. These 24 food samples were the first samples used in experiments.
To examine the quality variation of the products, the same products from the same shop werepurchased in the following week. In total, 48 samples were collected and used for the experiments.
To evaluate the quality of pickle products, Brix, salt concentration, and pH value were measured
for the pickle products. Three samples were more than three times different in Brix values between
the samples purchased on the first versus second weeks. Seven samples were more than 1% different
in salt concentration values. Three samples varied in pH values greater than 1.0. The fact that thedifferent values were obtained between the same pickle products showed the quality of pickle
products were unstable and/or the way of food processing was varied (data not shown).
Contamination and Spoilage Level of Foodborne Pathogen and Microbe in Pickle ProductsⒸ ISERD

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All samples were subjected to microbiological investigation. We investigated the generally viablemicrobes, coliform, fungi, Salmonella, S. aureus, and B. cereus to determine the food contaminationand spoilage levels. The total number of generally viable microbes of 48 samples is shown in Table
2. Fifteen samples were over 7 log10 CFU/g, indicating initial putrefaction. The samples from market
B and C had relatively higher total CFU than those from market A. The sample C, pickled young
onion, sample D and E, and pickled mustard showed a higher number (6 log10 to 8 log10) of CFU(data are not shown).
Table 2 Total number of general viable microbes in pickle products
Marketlog10
CFU/gABC<31stsample
1

2ndsample
1

1stsample
1

2ndsample
2

1stsample
0

2ndsample
1

4

2

1

100

1

5

3

3

2

1

1

1

60

2

3

1

3

2

7

1

1

1

32

2

8

1

0

0

1

2

1

Next, we investigated major food spoilage microbes and food pathogens. Results are shown in
Table 3. Coliform is an indicator of whether or not the food was handled in a proper manner. Coliform
inhabit the human intestinal tract, the presence of them means likely fecal contamination (Madigan,2019). As a result, the total contamination rate of coliform was 58.3%. According to Ceuppens (2014),
the existence of coliform was correlated to the presence of E. coli and a sufficient amount of E. coli.
More than half of the pickles were coliform positive and had a significant risk of including pathogenic
E. coli such as EHEC.
Microbial spoilage of fresh vegetables is often caused by fungi (Madigan, 2019) that are able tosurvive at lower aw conditions. So, the contamination by fungi was examined. The total rate ofcontamination was 68.8%.
Salmonellosis is very common for bacterial food infection. This disease is typically caused bySalmonella and Salmonella-contaminated food and animals. Of the first 24 samples, two samples
were Salmonella positive and in the second batch of samples, 18 samples were positive. In total,
41.7% of pickles were contaminated by Salmonella. Combined with the wet market conditions shownin our previous study, Salmonella contamination was most likely caused by cross-contamination ofchicken and egg products and by Salmonella-carrying animals such as dogs that come into contact
with the food.
S. aureus cause food poisoning and the produced toxins by S. aureus cause gastrointestinal
symptoms. These toxins can be superantigens that lead to lethal toxic shock syndrome. The presenceof S. aureus (83.3%) was surprisingly high in our tested samples. Chrun et al. (2017) reported thatthe contamination rate of fermented vegetables collected from the wet market in Phnom Penh wasless than 10%, but the samples used in this study exceeded 80%. S. aureus can grow in both aerobic
and anaerobic conditions and in many foods. S. aureus is often carried by humans. The interior ofnostrils and skin are associated with S. aureus and a pus-forming wound becomes a frequent cause(Madigan, 2019; Matthews, 2017). In our previous study, we observed that the vendors handled theirproducts with bare hands and utensils that were left out. Most staphylococcal contamination is causedby humans during food preparation (Matthews, 2017). It is assumed that the inappropriate manner offood handling increases the chance of food contamination.
B. cereus is widespread in nature and is frequently isolated from growing plants and soil. So,
this organism is easily spread if there is no equipment to prevent dust and dirt from the outside.
Foodborne illness by B. cereus is of two types—the emetic form results in vomiting and the diarrheal
form results in diarrhea. Our results showed that 37.5% of the total samples were B. cereus positive.
Upon microbiological examination, there were large variations in between different pickle productsⒸ ISERD124

IJERD – International Journal of Environmental and Rural Development (2020) 11-1and even within the same pickle products. Some pickles were considered to contain enough sugar andsalt to prevent spoilage; however, most products had some kind of microbiological hazard. Our
previous study revealed that the wet market was in a condition where cross-contamination can easily
occur. The cause of bacterial contamination could be from raw materials and cross-contamination. The
water used for food processing and cleaning may also be a source of foodborne pathogens (Steel, 2004).
Table 3 The rate of microbiological contamination in pickle products
Number of positive / Number of total
Microbial sp.
First sample

Second sample

Total rate(%)

Coliform

14/ 2414 /24

58.3

Fungi
Salmonella

13 / 24

20 / 2468.8

2 / 2418 / 24

41.7

S. aureus

19 / 24

21 / 2483.3

B. cereus

12 / 24

6 / 24

37.5

CONCLUSION
In this study, we investigated the level of food spoilage and the rate of food contamination of pickle
products. The results indicated that significantly poor sanitary conditions and unsafe food products
were provided at the wholesale market in PP. It is necessary that food handlers as well as consumers
receive practical knowledge on food hygiene to prevent the spread of bacterial food contamination.
In the future, we will focus on the cause of food contamination and give countermeasures against it.
ACKNOWLEDGEMENTS
The measurements of food properties and microbial examination were conducted at the Faculty ofAgro-Industry, Royal University of Agriculture, Cambodia.
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