Sensitivity of enrichment-PCR method for Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis analysis in chicken carcasses

Salmonella spp. is Gram negative-pathogenic bacteria that usually found as a contaminant in chicken carcasses. This study was aimed to increase the sensitivity of PCR enrichment step and apply the enrichment-PCR combination to detect Salmonella in chicken carcasses. In this study were used Salmonella enterica serovar Hadar, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis with the target genes were invA, STM4497, and respectively. A total of 25 g of the chicken carcasses were artificially contaminated by approximately 0.96 and 3.33 MPN/mL for each serovar separately. Samples were incubated in pre-enrichment and enrichment media for 8 hrs prior to the DNA extraction. The pre-enrichment and enrichment media was Buffered Peptone Water and Rappaport-Vassiliadis-soya. The result showed that the target genes of S. enterica ser. Hadar, S. enterica ser. Typhimurium and S. enterica ser. Enteritidis were detected in chicken carcasses, indicated by the presence of DNA band with the size was 429 bp, 311 bp and 135 bp respectively. These result in line with analysis using ISO method and BLAST-comparison analysis of DNA amplicon sequences with GenBank references. Application of this method for Salmonella detection in chicken carcasses sold in the traditional market showed a higher prevalence than the previous result without enrichment. All samples (n = 100) from unsanitary practice sellers were positively contaminated by Salmonella spp. and also high prevalence for S. enterica ser. Typhimurium and S. enterica ser. Enteritidis. It can be concluded that enrichment is an important step to increase the sensitivity detection of PCR method.


Introduction
Salmonella spp. is facultative anaerobic bacteria, Gram-negative, and rod-shaped bacilli. This bacterium belongs to family Enterobacteriaceae (Kim et al., 2006). More than 2600 serovars Salmonella spp. have been identified (Popoff et al., 2003). Salmonella has been known as a foodborne pathogen and caused outbreaks in several countries. Salmonella infections occurred in America (Scallan et al., 2011), China (Xiao et al., 2015), Australia (Ford et al., 2018), Canada (Morton et al., 2019). Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis are the dominant contaminant serovars in chicken carcasses. Both serovars are reported being the main cause of salmonellosis in humans ). Molecular detection is proposed to be an effective and reliable method to detect Salmonella spp. in this research. The basic principle of the PCR method is to amplify the DNA fragment of target bacteria (Joshi and Deshpande, 2011). The PCR method has widely used to detect Salmonella in food (Wang et al., 2018). The invA gene is responsible for the virulence of Salmonella spp. and can be found in all serovar Salmonella spp. (Shanmugasamy et al., 2011). The STM gene encodes fimbrial biosynthesis, which found in S. enterica ser. Typhimurium, particularly (Clavijo et al., 2006). Fimbrial in S. enterica ser. Typhimurium has a function for adhesion, attachment of bacteria to the target cell, and start the infection (Malorny et al., 2003). The Prot6E gene is 60 kb virulence gene and only found in in S. enterica ser. Enteritidis (Malorny et al., 2007).
Previous developed molecular detection has low sensitivity, so it needs to be combined with the enrichment techniques (Yosua, 2018

Materials
The materials used in this study were in S. enterica ser. Hadar (BCC B2908), in S. enterica ser. Enteritidis (ATCC 13076)

DNA extraction
The DNA extraction of the deposition method by chelex100 resin was performed as described by Reyes et al. (2010). Salmonella bacterial culture was centrifuged for 20 mins at 3000 rpm to separate the bacterial cells. The formed sediment or bacterial cells were transferred into the microtube and recentrifuged for 5 mins at 8000 rpm at 4 o C. The pellet that settles at the bottom of the tube was resuspended with a 1000 µL TE buffer solution (10mM TRIS-base pH 7.5; 1 mM EDTA pH 8.0). The mixture of the solution was centrifuged for 5 mins at 8000 rpm at 4 o C. The pellet was resuspended with 100 µL TES buffer lysis (10 mM TRIS-base pH 7.5; 1 mM EDTA pH 8.0; 0.5% SDS), incubated at 65 o C for 5 mins. Approximately, 7.5 μL proteinase and 2 μL RNAse were added to the solution, incubated at 65 o C for 5 mins, and at room temperature for 2 mins. A total of 150 µL of chelex100 TE buffer was added into the tube, incubated at 65 o C for 5 mins, centrifuged for 10 mins at 10000 rpm at 4 o C. The aqueous phase of the solution was transferred into a clean microtube. 3M sodium acetate was added as much as 0.1 times the volume of the liquid layer, and 95% ethanol 2.5 times of the volume. The mixture was incubated in the freezer at -20 o C for 20 hrs. After the centrifugation of the mixture for 5 mins at 8000 rpm, the supernatant was removed, and the tube containing the pellet at the bottom of the tube was dried at room temperature (26-27 o C). Approximately, 75 µL of NFW was added into a tube containing the dry pellet and stored in a freezer for further analysis.

PCR amplification and gel electrophoresis
The mixture for the PCR reaction was 10 µL Master Mix (GeneAid), 1 µL of each primer forward and reverse, 7 µL Nuclease Free Water, and 1 µL DNA template. The PCR instrument was set for an initial denaturation at 95°C for 3 mins for 1 cycle, followed by denaturation temperature at 95°C for 1 mins for 35 cycles, annealing temperature at 52°C for 1 mins and extension temperature at 72°C for 1 mins, followed by a final extension temperature at 72°C for 5 mins. The running condition of the PCR instrument was set according to the Ta (Temperature annealing) and Tm (Temperature Melting) of a primer so the amplification process could occur properly. The primers and gene targets are detailed in Table 1. The PCR products were separated using electrophoresis gel for 45 mins at 90 V. The visualization was done using Gel Documentation after the gel was stained in EtBr for 30 mins.

Enhancement of sensitivity and Salmonella spp. detection using enrichment technique
A total of 25 g of the chicken carcasses were put in a Approximately, 1 mL pure cultured of each S. enterica serovars was added into plastic bags separately with 1 to 10 CFU/mL. Then, 225 mL of BPW media was added to the plastic bag containing the sample and bacterial culture and incubated for 2, 4, 6, and 8 hrs at 37 o C. The fastest incubation time was chosen with a 100% positive percentage, then it was combined with another incubation using RVS media. An aliquot (1 mL) of the obtained culture was transferred to 10 mL RVS then incubated at 42 o C. The DNA was extracted from RVS media. Gel electrophoresis was used to visualize the PCR product. The analysis was carried out five times for each serovar to check the stability of this method. It also needs to be confirmed using a standard ISO 6579:2002 method. Confirmation of the PCR products was done by DNA sequencing.

Confirmation by ISO 6579: 2002 and DNA sequencing
The confirmation by ISO 6579:2002 method was performed using ten chicken carcasses samples. A total of 25 g of the sample was put into a sterile plastic bag containing 225 mL BPW and was incubated for 18 hrs at 37 o C. As much as 0.1 mL of suspension was transferred into a tube containing 10 mL RVS media, and was incubated for 18 hrs at 42 o C. A loopful of RVS suspension was streaked aseptically onto XLDA and TSIA slant and the slants were incubated for 18 hrs (ISO, 2002).
PCR products that showed positive result of each gene of invA, STM4497, and Prot6E were sent to First BASE Laboratories in Malaysia for DNA sequencing. Sequencing results were analyzed using the Bioedit 7.2.5 program (http://bioedit.software.informer.com/7.2/). Then, the results were matched with data in the GenBank data center via the NCBI website (https:// www.ncbi.nlm.nih.gov/) using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.Cgi ).

Application of confirmed methods in chicken carcasses
The enrichment-PCR method that had been confirmed in the previous stages was used in detecting Salmonella spp. in chicken carcasses. The sample was 100 chicken carcasses from nineteen traditional markets in Bogor, Indonesia. The selected sellers have not applied good sanitary practices for handling their products based on previous observations.

Enhancement of sensitivity and Salmonella spp. detection using enrichment technique
All three serovars Salmonella spp. could be detected at very low bacterial counts (0.96-3.33 MPN/mL) with incubation times from 4 to 8 hrs on BPW media ( Table  2). The incubation time of 2 hrs was not enough to detect these bacteria at a low number. Therefore, the incubation time for 4 hrs was chosen. The number of Salmonella spp. was increased from 10 2 to 10 8 CFU/mL, it was detected using the fibre-optic method with a combination of enrichment in BPW and RV for 4 hrs incubation in each media (Abdelhaseib et al., 2016). The log number of in S. enterica ser. Enteritidis and in S. enterica ser. Typhimurium were increased from 10 to 10 5 CFU/mL with an incubation time of 8 hrs on BPW (Josefsen et al., 2007).
BPW used as non-selective pre-enrichment media and RVS used as selective enrichment media. In nonselective media, the injured cells of both pathogen bacteria target and other bacteria are usually repaired (Chen et al., 2013). RVS media should promote the growth of the recovered bacteria target (Margot et al., 2015). RVS has more nutrients resulting in better growth of individual Salmonella strains (Gorski, 2012). RVS components such as MgCl 2 will increase osmotic pressure along with malachite green and low pH of this media inhibit the growth of non-Salmonella (Rappaport et al., 1956;Vassiliadis et al., 1978). The enrichment-PCR analysis with a combination of BPW and RVS for a total of 8 hrs gave a 100% positive percentage in detecting Salmonella spp. with low detection limit (Table  3).  (Gorski et al., 2011). TSIA contained lactose, sucrose, and dextrose used to detect bacteria that can ferment those sugars and produce H2S (Majeed, 2016). The result shows all samples (n = 10) were positively contaminated with Salmonella spp. not only with the enrichment-PCR but also with the ISO 6579:2002 method. Positive results were marked by the growth of black colonies on the XLDA plate. Salmonella spp. is a bacteria that can ferment sugars, so the positive result was marked by a change in color and the formation of gas in TSIA slant.

Confirmation
Another confirmation was performed by sequence of PCR product (amplicon) of 2 positives samples for Salmonella spp., in S. enterica ser. Typhimurium, and in S. enterica ser. Enteritidis with invA, STM4497, and Prot6E primer respectively. The results were analyzed using BioEdit to find consensus region of the base sequence before further analysis with BLAST NCBI (Table 4). S. enterica ser. Typhimurium and S. enterica ser. Enteritidis appeared in BLAST analysis of sequenced samples (Table 5). The BLAST analysis of the sequenced sample with invA primer showed that there were S. enterica ser. Enteritidis and S. enterica ser. Typhimurium as serovar matched with the genebank database. The invA gene located in a chromosome known as Salmonella Pathogenicity Island (SPI) I, a protein encoder, in the inner membrane of bacteria (Boddicker et al., 2013). The majority of Salmonella virulence genes distributed in a chromosome called SPI (Pal et al., 2017). The invA gene is responsible for the virulence of Salmonella spp. and found in all serovar Salmonella spp. (Shanmugasamy et al., 2011). Several bacteria genus were detected simultaneously by the multiplex PCR method. The invA gene was used as a gene target for the detection of Salmonella spp. (Kim et al., 2007). The identification of Salmonella spp. in poultry meat was carried out using invA primers (Sharma and Kashmiri, 2016). Compared to other virulence genes, invA gene was detected in all Salmonella isolates (Thung et al., 2018).
S. enterica ser. Typhimurium and Klebsiella pneumoniae appeared on the BLAST result. Further analysis was performed to confirm these findings. Based on the percent identity to sequenced samples, S. enterica ser. Typhimurium had 96.76%, while Klebsiella pneumoniae had lower percent identity, which was 96.46%. Alignment analysis conducted between the two bacteria and the sequenced sample also showed that the matched number of bases was higher in S. enterica ser. Typhimurium. The STM gene encodes fimbrial biosynthesis and only found in S. enterica ser. Typhimurium (Clavijo et al., 2006). Fimbrial in S. enterica ser. Typhimurium has a function for adhesion, attachment of bacteria to the target cell, and start the infection (Malorny et al., 2003).  Typhimurium, but has a genetic mutation (Ido et al., 2014). Salmonella 4,[5],12:i was the result of a genetic mutation of S. enterica ser. Typhimurium that lacks a second phase flagella antigen (Switt et al. 2009). The virulence gene in S. enterica ser. Typhimurium was found in serovar 4,[5],12:i to prove that the serovar belongs to the S. enterica ser. Typhimurium variant (Palma et al., 2018). Salmonella serovar Rough-O is a Typhimurium serovar that has an antigen-O deficiency in its genetic structure (Park et al., 2018).
The result of sequencing analysis with Prot6E primer showed that only S. enterica ser. Enteritidis in BLAST analysis from a total of 60 Ac. identified numbers. The enrichment-PCR method using Prot6E primers for the detection of S. enterica ser. Enteritidis obtained specific detection. The Prot6E is a 60kb virulent gene that only found in S. enterica ser. Enteritidis. Detection of Salmonella PCR tests other than S. enterica ser. Enteritidis showed negative results using the Prot6E gene target (Malorny et al., 2007).

Application of confirmed methods in chicken carcasses
Chicken carcasses sample were obtained from several traders in traditional markets in Bogor region, Indonesia. The selection of traditional markets was based on their poor sanitation of facilities and sanitary behaviour of the traders. Most of the traders stored chicken carcasses outdoors and did not store in refrigerator temperatures. Samples were whole chicken carcasses, not frozen, and estimated it had been on display for 2-5 hrs. The result of the analysis using the enrichment-PCR method shows a high prevalence of contamination. In contrast, previous studies using the non-enrichment method showed 60% contamination for Salmonella spp., 16% for S. enterica ser. Typhimurium and 60% for S. enterica ser. Enteritidis (Melati, 2019

Conclusion
Enrichment for 4 hrs on non-specific pre-enrichment media and 4 hrs on specific enrichment media can increase the sensitivity of PCR detection. Confirmation of sequenced amplicon to Genebank indicated the specific primer sequences used, invA gene for the Salmonella genus (429 bp), STM4497 for S. enterica ser. Typhimurium (311 bp) and Prot6E (135 bp) for S. enterica ser. Enteritidis, are specific primers referring to those serovars. This method can be applied to detect contamination in chicken carcasses by showing a high prevalence of findings.