Posted on in the International Journal of Philippine Science and Technology 2015 Volume 8 Issue Number 2 Page 52—55
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Submitted: May 15, 2015
Revised: August 7, 2015
Accepted: August 16, 2015
Published: September 30, 2015
Abstract—A simple and specific detection method for Salmonella enterica was applied to determine its incidence in raw and processed meats purchased from selected wet markets in Metro Manila, Philippines. A total of 320 raw and processed meat samples were analysed for the presence of S. enterica and S. enterica possessing spvC gene of the virulence plasmid. Polymerase chain reaction (PCR) revealed that 30.63% (98/320) were positive for S. enterica and 2.81% (9/98) of S. enterica-positive samples were also positive for the spvC gene. S. enterica was identified from chicken samples (67.5%), ground pork (65%), beef (52.5%), sausage (longganisa) (25%), cured pork meat (tocino) (20%), burger patty (12.5%), and meatloaf (embotido) (2.5%). Positive samples were further analysed for O-serogrouping targeting S. enterica serogroups A, B, C1, C2, D, and E1. Our findings revealed that the raw and processed meats tested were contaminated with more than one serogroup in a sample. Samples were found positive for S. enterica serogroups E1 (78.57%), C1 (29.59%), C2 (20.41%), B (17.35%) and D (6.1%). No samples were found positive for S. enterica serogroup A. This is the first report on the use of multiplex PCR for the detection and characterization of S. enterica in raw and processed meats in the Philippines. Data on incidence of S. enterica and its serogroups found in raw and processed meats in selected Philippine wet markets gathered from this study can be used for further research on epidemiology and related topics.
Keywords—Salmonella enterica, serogroups, spvC gene, raw and processed meats, wet markets, Metro Manila
Potential biological hazards in meat include bacteria, toxins, viruses, protozoa and parasites. Of these, bacteria cause a large proportion (approximately 90%) of all foodborne illnesses. The bacterial pathogens that are most likely to be found in commonly slaughtered livestock (cattle, sheep, and swine) and poultry (chicken and turkey) include Salmonella enterica, Campylobacter sp., and Listeria monocytogenes. These pathogens have been implicated in widely publicized foodborne disease outbreaks associated with the consumption of meat and poultry products (Food Safety and Inspection Service 1999). Salmonellosis, which is caused by Salmonella, is one of the leading causes of foodborne bacterial enteritis in many countries (Centers for Disease Control and Prevention 2004a; Tirado and Schmidt 2001). Outbreaks of diseases are reported frequently, implicating different kinds of food contaminated with S. enterica (Centers for Disease Control and Prevention 2004b; Ethelberg et al. 2004; Fielding et al. 2003; Matsui et al. 2004). S. enterica is a common cause of human gastroenteritis and bacteremia worldwide (Hendriksen et al. 2009; Morpeth et al. 2009; Schlundtet al. 2004; Voetsch et al. 2004). A wide variety of animals, particularly food animals, have been identified as reservoirs of non-typhoidal Salmonella (Bangtrakulnonth et al. 2004; Guard-Petter 2001; Vindigni et al. 2007). Currently, Salmonella consists of 2,659 different serovars (Issenhuth-Jeanjean et. al. 2014) and based on the Salmonella Serotype Statistics, approximately 60% belong to subspecies I, S. enterica subsp. Enterica (Centers for Disease Control and Prevention 2008).
Raw and processed meats in the Philippines are typically sold in two types of markets, supermarkets and wet markets. The supermarkets, which are indoor markets, often display pre-packaged raw meat under refrigeration. In contrast, the wet markets usually display unwrapped raw and processed meats at ambient temperatures which are easily contaminated with foodborne pathogens. Reports of S. enterica contamination on raw and processed meats in the country are limited. Reliable detection methods are therefore required for diagnosis and prevention of food contamination and foodborne outbreaks. Traditional microbiological techniques, such as the International Organization for Standardization (ISO) method 6579 for detecting Salmonella in food, take up to 5 days to obtain a positive result. This ISO method includes pre-enrichment and selective enrichment in liquid culture, and biochemical and serological confirmation of colonies grown on agar plates (International Organization for Standardization 2002). Delays caused by the identification procedure can hinder the appropriate response to an outbreak of disease. For a more reliable and fast analysis, polymerase chain reaction (PCR) has been applied in various stages of the diagnostic procedure: confirmation of suspected colonies grown on agar plates, analysis of enrichment broths, and direct analysis of suspected foodstuffs. PCR employs specific primers to detect the presence of pathogens in a shorter period of time. Various studies used this technique for the detection of Salmonella spp. (Chiu and Ou 1996; Kawasaki et al. 2009; Pritchett et al. 2000). Furthermore, studies on the application of molecular methods in detection and characterization of S. enterica isolates in the Philippines are limited. Multiplex PCR of S. enterica isolates, for instance, is not widely performed in the Philippines. As such, known serogroups are not accurately documented.
This study aimed to determine the presence of S. enterica in raw and processed meats from four wet markets in Metro Manila, Philippines: Commonwealth, Balintawak, Cubao, and Caloocan. Samples included raw meat such as cut chicken, minced pork, cut beef and processed meats, such as homemade meatloaf (locally called embotido) , sausage (locally called longanisa), cured pork meat (locally called tocino) , burger patty, and ham. Specifically, this study aimed to: (1) detect S. enterica via multiplex PCR targeting the invA and spvC genes; and (2) characterize S. enterica isolates via multiplex PCR detection of the somatic (O) antigen.
Three hundred twenty (320) raw and processed meat samples were randomly obtained from selected wet markets in Metro Manila, Philippines: Balintawak, Caloocan, Commonwealth, and Cubao (Table 1). Ten samples of each type of meat were collected in each of the four wet markets. All samples were placed in a cooler (approximately 5 °C) during transport and immediately processed in the laboratory for pre-enrichment. Samples were labeled according to their location, sample type and numbered from 1 to 10.
Table 1. Incidence of Salmonella enterica in raw and processed meat samples collected in selected wet markets in Metro Manila, Philippines.
|Meat Sample||Type of Meat||Code||Number of samples per site||Number of samples in 4 wet markets||Number of culture-positive samples||Number of PCR-positive samples|
To determine the presence of S. enterica in the samples, conventional culture method was used. Twenty five (25) grams of sample was suspended into 225 ml buffered peptone water (BPW, Difco) and incubated at 37 °C for 18-24 h. One hundred microliters of pre-enriched culture was simultaneously transferred to 10 ml Rappaport Vassiliadis Soy enrichment broth (RVS, Merck) and incubated at 42 °C for 18-24 h. Enriched RVS culture was inoculated in xylose lysine deoxycholate agar (XLD, Merck) and brilliant green agar (BGA, Difco), and then incubated at 37 °C for 24-48 h at aerophilic condition. Growth on either BGA or XLD was considered culture-positive. S. enterica serovar Typhimurium UPCC 1360 obtained from the University of the Philippines-Culture Collection (UPCC) in the Microbiological Research and Services Laboratory (MRSL) of the Natural Sciences Research Institute (NSRI), was used as positive control.
In each sample, 500 µl of the RVS-enriched culture was collected from triplicate tubes into a microfuge tube and centrifuged at 15,330 × g for 5 min. The supernatant was discarded, and the cell pellets were suspended in 1 ml phosphate buffered saline (PBS), centrifuged at 15,330 × g for 5 min. The supernatant was again discarded, and the pellets were suspended in 50 μl sterile distilled water for DNA extraction (O’Regan et al. 2008). The suspension was heated at 99 ºC for 10 min (Shanmugasamy et al., 2011), and then pelleted by centrifugation at 2,656 × g for 5 min. The DNA extracts were immediately cooled on ice.
DNA amplification was performed in a reaction volume of 25 μl. Each reaction mixture contained 12.5 μl of 2× GoTaq® Green Master Mix (Promega), 1 μl each of 10 μM forward and reverse primers of invA and spvC genes (Table 2), 7 μl of sterile double distilled water, and 1.5 μl DNA template extracted from RVS broth. PCR was performed under the following conditions: initial denaturation at 95 °C for 2 min, denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s and final extension at 72 °C for 5 min. Amplicons were visualized by a UV transilluminator (BioRad®) after electrophoresis on 1.5% agarose gel stained with ethidium bromide (0.5 µg/ml). The Salmonella Typhimurium UPCC 1360 obtained from MRSL was used as positive control, while sterile double distilled water was used as negative control.
Table 2. Primer sequences used for multiplex PCR methods for the detection of Salmonella enterica.
|Primer||Sequence (5'-3')||Amplification target||Amplification length (bp)||Tm (°C)||Reference|
|F-invA||ACAGTGCTCGTTTACGACCTGAAT||S. enterica||244||50.6||Chiu and Ou 1996|
|F-spvC||ACTCCTTGCACAACCAAATGCGGA||S. enterica plasmid virulence||571||65.6||Chiu and Ou 1996|
|F-abe1||GGCTTCCGGCTTTATTGG||B group||561||45.2||Hong et al. 2008|
|F-tyv||GAGGAAGGGAAATGAAGCTTTT||D group||614||46.0||Hirose et al. 2002|
|F-prt||CTTGCTATGGAAGACATAACGAACC||A & D group||256||50.9||Hirose et al.2002|
|F-wbaD-manC||ATTTGCCCAGTTCGGTTTG||C1 group||341||43.8||Hong et al. 2008|
|F-abe2||CGTCCTATAACCGAGCCAAC||C2 group||397||48.7||Hong et al. 2008|
|F-wzx–wzy||GATAGCAACGTTCGGAAATTC||E1 group||281||45.3||Hong et al. 2008|
DNA amplification was performed in a reaction volume of 25 μl. Each reaction mixture contained 12.5 μl of 2× KAPA2G Fast multiplex mix (KAPA Biosystems), 0.5 μl (0.2 μM each) of forward and reverse primers: prt, abe1, tyv, wzx-wzy, wbaD-manC, abe2 (Table 2), 3.5 μl of PCR grade water, and 2 μl DNA template. Multiplex PCR of the O-antigen was performed under the following cycling conditions: an initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 60 s. Final extension was done at 72 °C for 10 min. The positive control used is a mixture of DNA extracts consisting of Salmonella serogroups B (abe1-positive), C1 (wbaD-manC-positive), C2 (abe2-positive), D (tyv- and prt-positive), and E1 (wzx-wzy-positive), which were previously confirmed by DNA sequencing (Ng and Rivera, 2015), while sterile double distilled water was used as negative control.
For the invA-spvC assay, amplicons were visualized by a UV transilluminator (BioRad®) after electrophoresis (Mupid®-2 Plus) at 100 V for 25 min. These were run on 1.5% agarose gel stained with ethidium bromide (0.5 µg/ml). The sizes of the target sequences were estimated using a Universal DNA Ladder (KAPA Biosystems). The amplicons for the O-antigen assay were viewed similarly, but on 2% agarose gel and using a 100 bp DNA ladder (Vivantis) to estimate band sizes.
A total of 320 raw and processed meat samples were included for the detection of S. enterica. Ninety-six (96) out of 320 samples were culture-positive for S. enterica (30%), while PCR detection showed that 98 samples were positive for S. enterica (30.63%).
Notably, a higher incidence of S. enterica was found in raw meats compared to processed meats. A large percentage of chicken samples (67.5%) was positive for S. enterica, followed by ground pork (65%), beef (52.5%), longganisa (25%), tocino (20%), burger patty (12.5%), and embotido (2.5%). S. enterica was not detected in ham samples (Figure 1).
In this study, invA gene was present in 98 samples. The spvC gene was present in 2.8% of the S. enterica-positive samples. S. enterica possessing spvC was found in six chicken and three burger patty samples only.
The amplicons produced intense bands of different expected sizes, representing five targeted serogroups (Table 2). Of the 98 invA-positive S. enterica samples, 78.57% were positive for serogroup E1, followed by C1 (29.59%), C2 (20.41%), B (17.35%) and D (6.1%). There were no samples positive for serogroup A.
Figure 3. S. enterica O-serogroups (614-bp: tyv for D, 561-bp: abe1 for B, 397-bp: abe2 for C2, 341-bp: wbaD-manC for C1, 281-bp: wzx-wzy for E1, and 256-bp: prt for A and D). L –Vivantis VC 100bp DNA ladder, PC - positive control, NTC - no template control, 1-14 - isolates.
S. enterica was detected in raw and processed meat samples from selected wet markets using culture and multiplex PCR methods. These methods yielded high incidence of S. enterica, which supported the results of previous studies that showed the sensitivity of targeting the invA gene of S. enterica as a reliable detection method (Amini et al. 2010; Chiu and Ou 1996; Shanmugasamy et al. 2011). The invA gene has been recognized as international standard for detection of genus Salmonella. The expression of this gene enables Salmonella to invade the epithelial cells of its host (Shanmugasamy et al. 2011). Ninety-six (96) out of 320 samples were culture-positive for S. enterica (30%), while PCR detection showed 98 samples positive for S. enterica (30.63%). Nine samples harbored the virulence plasmid spvC gene, which reflects a relatively lower incidence than those in other published reports (Amini et al. 2010; Chiu and Ou 1996). Nevertheless, the presence of this gene indicates that these isolates may cause systemic disease (Heithoffet al. 2008).
The positive result for invA in two samples but negative for S. enterica in culture-based method suggests that the latter methods may overlook the presence and underestimate the number of S. enterica in a sample. El Shamy et. al. (2008) noted that conventional culture media may not be universally sensitive for the detection of Salmonella, depending on sample type or serotype.
The PCR-based detection used in this study is simpler, more rapid, and more specific for the detection of S. enterica compared to culture/traditional method of detection of S. enterica. The traditional method normally takes 7 days. With the multiplex PCR used in this study, identification of serogroups of S. enterica could be done in 3 days. Boiling method was selected for the preparation of the DNA template because of the convenience of the method. Centrifugation and washing steps were sufficient to remove the PCR inhibitors from the culture medium.
A large percentage of chicken (67.5%), ground pork (65%), and beef (52.5%) samples were positive for S. enterica. Several outbreaks and studies of salmonellosis in chicken/poultry have been reported (Rajagopal and Mini 2013; Wegener and Baggesen 1996). Ground meat (e.g. ground pork, burger patty) is among the food items frequently associated with outbreaks of salmonellosis. It is predisposed to contamination because many processing steps are involved in its manufacture (e.g. grinding), which potentially contribute to an increase of S. enterica counts in the final product (Stock and Stolle 2001). Thus, the incidence of S. enterica in ground pork could come from improper handling and use of unsanitary mechanical grinder in meat processing in wet markets.
S. enterica was also present in processed meat: longganisa (25%), tocino (20%), burger patty (12.5%), and embotido (2.5%), despite the presence of preservatives and additives (Table 1). Handling and transport were the most probable reasons for cross-contamination. The absence of S. enterica in ham samples could be attributed to thermal processing during the manufacturing process.
The highest incidence of 78.57% observed for S. enterica serogroup E1 was similar to the results of Balala et al. (2006) and Lee et al. (2009), which also showed the predominance of serogroup E1 among isolates from clinical and food samples collected in the Philippines. The multiplex PCR also detected S. enterica from serogroups C1 (29.59%), C2 (20.41%), B (17.35%) and D (6.1%). Four isolates were not characterized in multiplex PCR for O-serogrouping. Only six serogroups were targeted using the primers abe1, tyv, prt, wbaD-manC, abe2 and wzx-wzy in the O-serogrouping assays. Hence, these isolates could belong to other 40 Salmonella serogroups (Grimont and Weill 2007) that were not tested in this study.
Obtaining DNA extracts from the RVS-enriched medium allows for the detection of multiple S. enterica serogroups in one sample. It eliminates the need for a pure isolate of every serotype of S. enterica in a sample, thus saving time and effort. The multiplex PCR assay used for the serogrouping of the isolates may also be considered less expensive compared to conventional serotyping assuming that PCR and post-PCR equipment are available.
In this study, a simple and specific detection method for S. enterica was applied in raw and processed meat samples collected from selected wet markets in Metro Manila, Philippines. Through molecular characterization by targeting a portion of the invA and spvC genes, S. enterica isolates were detected and successfully characterized serologically via PCR-based detection of somatic (O) antigen. More than one serogroup were present in a sample; 78.57% (77 samples) with the predominance of S. enterica serogroup E1. The methods used in this study can greatly reduce the reliance on the costly and tedious conventional serotyping. They can be applied by any facility that lacks the expensive typing sera and expertise needed for conventional serotyping but is equipped with basic PCR facilities. Likewise, multiplex PCR can be useful especially during outbreaks when rapid detection is necessary.
This is the first report on the use of multiplex PCR for the detection and characterization of S. enterica in raw and processed meats in the Philippines. This study, likewise, was able to establish incidence data for S. enterica and its serogroups found in raw and processed meats in selected Philippine wet markets which could be used for further research in epidemiology and related topics. The relatively high incidence of S. enterica in raw and processed meats illustrates the risk for Salmonella infection due to poorly processed meat.
We thank Phyllis Anne P. Paclibare, Pauline Dianne M. Santos, and Alyzza Marie B. Calayag for the technical assistance. This study was supported by research grants from the Department of Agriculture-Biotechnology Program Implementation Unit (Project Code DABIOTECH-R1212) and the Office of the Vice-Chancellor for Research and Development of the University of the Philippines Diliman (Project Code 151515 PNSE).
The authors declare no conflict of interests regarding the publication of this article.
SASDR and WLR conceptualized this study. The experiments were conducted by SASDR. SASDR and WLR prepared the manuscript.