A review of culture-dependent and molecular methods for detection of Salmonella in food safety

Email: upmtty@yahoo.com eISSN: 2550-2166 / © 2019 The Authors. Published by Rynnye Lyan Resources Food Research 3 (6) : 622 627 (December 2019) Journal homepage: http://www.myfoodresearch.com M IN I R E V IE W A review of culture-dependent and molecular methods for detection of Salmonella in food safety *Thung, T.Y., Lee, E., Wai, G.Y., Pui, C.F., Kuan, C.H., Premarathne, J.M.K.J.K., Nurzafirah, M., Tan, C.W., Malcolm, T.T.H., Ramzi, O.S.B., Wendy, D.R., New, C.Y. and Son, R. Department of Food Science, Faculty of Food Safety 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 Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abdin, 22200 Kuala Besut, Terengganu, Malaysia Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia Department of Agricultural and Food Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Perak, Malaysia Department of Livestock and Avian Science, Faculty of Livestock, Fisheries and Nutrition, Wayamba University of Sri Lanka, Makandara, 60170 Gonawila, Sri Lanka Department of Diagnostic and Allied Science, Faculty of Health and Life Science, Management and Science University, 40100 Shah Alam, Selangor, Malaysia


Introduction
Foodborne infectious disease caused by bacterial pathogens is a major health problem worldwide.Among the foodborne pathogens, Salmonella is the most common bacteria associated with outbreaks known as salmonellosis, and is contracted through consumption of contaminated animal products, which always results either from infected animals used in food production or from contamination of the carcasses (Cheung and Kam, 2012;Park et al., 2014).Based on surveillance studies, the main vehicles of Salmonella infection include raw meat, eggs and poultry products (de Freitas et al., 2010;Sallam et al., 2014).Of greater consequence is that fruits and vegetables have gained notoriety in recent years as vehicles of human salmonellosis (Mritunjay and Kumar, 2015).The prevailing hygienic conditions especially in tropical and subtropical countries during the production, harvesting and distribution of fresh and dehydrated fruits and vegetables do not always meet minimum standards and may facilitate product contamination.For example, Salmonella spp.and Salmonella enterica serotype Typhimurium were detected in sliced fruits, like papaya, mango, jackfruit, watermelon, sapodilla, honeydew and dragon fruit (Pui et al., 2011), and local salad known as ulam (e.g., selom, pegaga nyonya, kacang panjang and kacang botol) (Najwa et al., 2015).
Rapid and reliable detection methods such as standard culture methods coupled with molecular techniques are necessary to control the safety of food.Typically, rapid methods have become increasingly popular among food testing laboratories and some of them have been accepted by international authorities as standard methods after thorough evaluation and validation.Therefore, different aspects of Salmonella detection and identification methodologies will be discussed in this review article.

Detection and enumeration of Salmonella
Detection and enumeration of Salmonella in foods and on surfaces that come into contact with food are an important component for ensuring food safety.For instance, conventional detection methods which provide both qualitative and quantitative information are used to isolate and enumerate viable bacterial cells in foods (de Boer and Beumer, 1999).Recently, considerable efforts have been made to improve the detection of Salmonella in different food ingredients by employing molecular detection method (Cheung and Kam, 2012).Hence, microbiological analysis comprises methods for quantitative detection is often used by both government authorities and food companies to monitor the state of contamination as well as to detect emerging risks (Park et al., 2014).

Conventional methods
Conventional culture methods acquired good reputation as standard techniques due to their selectivity and sensitivity (Taskila et al., 2012).Moreover, conventional test methods are simple, very practical and easily adaptable.Based on the standard ISO 6579:2002, detection process was involved in four stages: (a) nonselective pre-enrichment, (b) selective enrichment, (c) plating on selective media, and (d) biochemical and serological tests.
Prior to the enrichment cultivation, Salmonella could be injured during handling procedures and/or food processing, such as freezing, refrigeration, irradiation, drying, thermal treatment, low water activity, acidity, and starvation (Gracias and McKillip, 2004;Shintani, 2006).Thus, the first-line recovery of injured cells needs to be performed in a non-selective pre-enrichment step during the initial analysis.Buffered peptone water (BPW) contains sodium chloride and phosphates for the purpose of ensuring buffering capacity and maintaining osmotic balance, respectively.Due to low amounts of the target bacterial cells present in mixed microbial population, BPW often used to increase it (Taskila et al., 2012).Since non-selective media contain peptones (main sources of nitrogen and carbon), it might support or enhance other microbes to grow.Therefore, variety selective enrichment broths such as selenite cystine broth (SC), tetrathionate broth (TT), Rappaport Vassiliadis soy broth (RVS) and others have been developed for Salmonella isolation purposes (Taskila et al., 2012).RVS was reported as the best selective enrichment broth for Salmonella cultivation where it contains high concentration of malachite green and magnesium chloride which could be used to inhibit the growth of other microbial (Schönenbrücher et al., 2008).
Selective agar media such as brilliant green (BG) agar, bismuth sulphite (BS) agar and xylose lysine desoxycholate (XLD) agar are commonly used to differentiate and to receive presumptive Salmonella colonies (Sallam et al., 2014).Previously, chromogenic media such as CHROMagar Salmonella, chromogenic Salmonella esterase (CSE) agar, and alpha-beta chromogenic medium (ABC medium) have been developed to improve the selectivity (Perry and Freydière, 2007).In another study, researchers found that several chromogenic plating media such as Miller-Mallinson agar (MM), AES Salmonella agar plate (ASAP) and oxoid Salmonella chromogen media (OSCM) did not show any detection problems for falsepositive results in contrast to BG, XLD and xylose lysine tergitol 4 (XLT4) agar (Schönenbrücher et al., 2008).
Preliminary identification of Salmonella is often performed by biochemical tests, which may include the negative urease reaction, H 2 S production, fermentation of dulcitol and glucose (Mirmomeni et al., 2009), whereas confirmation of the Salmonella genus is determined by serological tests using somatic (O), flagellar (H) and capsular (Vi) antigens and their corresponding antibodies (Sallam et al., 2014).For instance, identification via biochemically and serologically was reported on various Salmonella serovars such as Agona, Arizonae, Bardo, Cerro, Dublin, Hadar, Gallinarum, Kentucky, Paratyphi A, Enteritidis, Typhimurium, Heidelberg and Virginia (Moussa et al., 2012).
On the other hand, the most probable number (MPN) method is widely applied to enumerate low concentration of microorganisms with <10-100 MPN/g (Malorny et al., 2008).It appears particularly interesting as it provides quantitative data, ideally for estimating the numbers of viable bacteria which are distributed randomly within the sample.For example, researchers had employed MPN method to enumerate S. enterica serovar Typhimurium and S. enterica serovar Typhi in sliced fruits with estimated quantity ranging from 0 to 19 MPN/g (Pui et al., 2011).Similarly, MPN method was used to determine the density of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium concentration in retail beef (Thung et al., 2018) and chicken meat (Thung et al., 2016).Recently, Tan et al. (2019)

Molecular detection methods
The advent of nucleic acid-based technologies, which referred to as rapid method, has improved sensitivity and specificity for detecting foodborne Salmonella with enhanced analytical performance and testing efficiency (Cheung and Kam, 2012).Molecular techniques are able to detect the target pathogens until defining subtle genetic differences at the strain level although the pathogens present in complex biological environments (Park et al., 2014).Therefore, these rapid detection methods could be of high value to the food industry.

Polymerase chain reaction (PCR)
Since the 1990s, molecular-based PCR assays are commonly used for the detection of Salmonella pure isolates from different samples (Soumet et al., 1999;Abouzeed et al., 2000).For instance, PCR assays have been used to detect selected virulence or antibiotic resistance genes along with genus-, species-, and/or serotype-specific genes of Salmonella in poultry and poultry products (Maciorowski et al., 2000;Oliveira et al., 2002).PCR methods employ several primer pairs which specific to Salmonella genes, resulting in high sensitivity for the detection of Salmonella (Table 1).
The essential for detecting low levels of Salmonella and the non-even distribution of the bacterial cells in the samples, an enrichment step is usually required prior to PCR assay.It has been proposed that the use of culture enrichment prior to PCR analysis serves the following purposes, (i) multiplication of the target bacterial cells to provide detectable concentrations, (ii) dilution of dead cells, (iii) dilution of PCR-inhibitory substances such as detergents, organic and inorganic chemicals, polysaccharides, fats and denatured proteins present in the sample matrix, and (iv) the possibility of isolating the target bacterial cells for complementary tests (Cheung and Kam, 2012).For instance, PCR assay failed to detect positive samples when no pre-enrichment step was carried out for the detection of Salmonella in raw poultry tissue samples of naturally contaminated (Myint et al., 2006), but Salmonella was successfully detected after the enrichment cultivation step.In another study, a PCR assay incorporated with several DNA extraction methods was used for Salmonella detection in poultry samples (Soumet et al., 1994).They found that the presence of Salmonella was detected using PCR associated with an enrichment cultivation step for 10 hrs.Besides, detection of Salmonella at different enrichment times in poultry dietary samples using PCR was also investigated by Maciorowski et al. (2000).Noteworthy, one of the major factors affecting the efficiency of PCR is depending on the quality of the target DNA.However, the presence of contaminated DNA had resulted in poor efficiency of the PCR (Wegener et al., 2003).Therefore, developments of other sample preparation methods based on the sample matrix type are indispensable to improve PCR detection.

Multiplex PCR (mPCR)
The mPCR is a simultaneous detection for multiple targets DNA sequence occurs within a single reaction.It has been widely used for simultaneous detection of different pathogens or virulence factors, as well as applied in nucleic acid diagnostics field, which includes gene deletion and mutation analysis (Xu et al., 2013).Also, mPCR was used for screening of single nucleotide polymorphism (SNP) (Hayden et al., 2008).High compatibility of the primers and the reaction mixture is of great significance for mPCR, especially in the detection of Salmonella simultaneously with other pathogens in food samples.For example, four sets of specific primers were used in mPCR to detect Escherichia coli O157: H7, Salmonella spp., Staphylococcus aureus, and Listeria monocytogenes in kimchi products (Park et al., 2006).Similarly, the detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. in meat and meat products, Vibrio parahaemolyticus and Vibrio cholerae in seafood and seafood products, and Staphylococcus aureus in ready-to-eat foods was also performed by using six pairs of primers in the mPCR assay (Lei et al., 2008).On the other hand, mPCR assay appears to be a more reliable method for identification of specific Salmonella serotypes, as compared to other assays by using phenotypic serotyping methods.In the study of Moussa et al. (2012)

Whole genome sequencing (WGS) of Salmonella
To date, WGS technology is used to evaluate and identify new species based on the entire DNA sequence of a bacterium thereby making it as a good surveillance tool (Ricke et al., 2018).WGS can be used to characterize individual microbes which include the full complement of resistance determinants, providing definite genotype information (McDermott et al., 2016).For example, WGS (Illumina HiSeq 2000 high throughput sequencing system) was used to analyze S. enterica serovar Weltevreden strains which isolated from contaminated food samples and human stool, where antimicrobial resistance (AMR) genes linked to resistance to ampicillin, ciprofloxacin or tetracycline was identified (Li et al., 2018).Previously, Wang et al. (2017) performed a WGS (PacBioRS II system) study analyzing Salmonella Indiana strain D90 isolated from the chicken carcass in China.They found that 24 multidrug resistance (MDR) genes, located on 4 plasmids, were identified, including the mcr-1 gene related to colistin resistance.Concurrently, a WGS approach was used to study cefoxitin-resistant Salmonella enterica serovar Heidelberg isolates from retail poultry, abattoir poultry and humans (Edirmanasinghe et al., 2017).They found that transmission of a common antimicrobial resistance plasmid (CMY-2) which linked to microbial resistance against β-lactamic antibiotics might occur among Salmonella Heidelberg strains with different genetic backgrounds.While the application of WGS coupled with specific computer programs might improve the interpretation of the heterogeneity typically in Salmonella genome classification.For instance, a software program Panseq was developed for the analyses of genomes as well as to identify bacterial strain (species -, subspecies-and serovar) using a SuperPhy platform (Laing et al., 2017).
have also described the use of MPN method in the enumeration of Salmonella spp. in raw beef from wet market and hypermarket.

Table 1 .
Sequences of oligonucleotide primers used for detection of Salmonella , mPCR was used for molecular typing of Salmonella enterica serovars, especially in detecting S. enterica serovar Enteritidis and S. enterica serovar Typhimurium.It has also been widely used for differentiation of the Enteritidis and Typhimurium serovars in meat samples by other researchers.For example, mPCR was successfully adapted to identify S.