Molecular detection by some virulence genes of Salmonella enterica subsp. enterica isolated from the stool of children with diarrhea

: Diarrhea is a real disease in childhood which could cause death. Therefore, this study was conducted to isolate Salmonella from 350 stool samples taken from children under five years in age, suffering from diarrhea during the period from March 2019 to March 2020 in Tikrit city / Iraq. The results showed the possibility to isolate ten isolates of Salmonella enterica subsp . Enterica, an infection rate, represents 2.875% of the total rate of patients who suffer from diarrhea. The virulence genes were investigated for ten isolates of S. enterica subsp . enterica , the result is that all isolates possessed the genes stn , invA , lpfA with an appearance percentage of 100%, while the percentage of appearance of pefA and fimH genes was 80% and 60%, respectively. All these isolates as Iraqi detection have been submitted to NCBI then accepted as ten Iraqi strains were in NCBI. All strains were concordance of 99-100% with the Salmonella enterica subsp. enterica Strains of Taiwan (CP085821.1), the United Kingdom (OU943338.1), China (CP085699.1), South Korea (CP077760.1), Pakistan (OK035700.1) and Hong Kong (CP0823381.1) in addition to four US states.


Introduction:
Salmonella is a foodborne pathogen that causes three clinical syndromes: typhoid fever, enteritis, and bacteremia.There are more than 2500 serotypes of Salmonella, which infect a variety of hosts.Nontyphoidal Salmonella (NTS) mainly causes gastroenteritis in humans, but it can cause acute bacteremia in young children and immunocompromised patients.It is estimated that around 3.4 million cases of bacteremia due to nontyphoidal Salmonella infection occur each year globally 1 .Reports indicate that 99% of Salmonella infections in humans are associated with serotypes of Salmonella enterica subsp.enterica 2 .The Salmonella genome contains several sets of genes, referred to as Salmonella pathogenicity islands (SPIs), that code for virulence factors.The genes encoding Salmonella virulence factors can be divided into two main classes, genes located on chromosomes such as the stn gene and genes located on the virulence plasmid 3 .Seventeen pathogenicity islands (SPIs) have been identified with Salmonella that contribute to its virulence, as well as many other genes such as Spv operon 4 .SPI-1 is believed to contain genes required for bacterial entry, while SPI-2 is essential for intracellular survival and systemic inflammation 5 .
The presence of virulence genes carried on a plasmid was first proposed in 1982, as the virulence plasmid in Salmonella spv (Salmonella plasmid virulence) is important in the process of Salmonella multiplying in the cells of the reticulo-endothelial system in warm-blooded vertebrates 6 .The invA gene contains unique sequences specifically for the Salmonella genus, it is located in the pathogenicity island (SPI-1), and is important for host epithelial cell invasion.This gene is present in most salmonella serotypes, so it is used as an important target for the detection of salmonella 7 .Long polar fimbriae (Lpf) is encoded by the lpfABCDE operon.These fimbriae are involved in the process of Salmonella adhesion to the surface of M cells in the Peyer's patches, which is the preferred entry port for Salmonella into intestinal epithelial cells 8 .The stn gene encodes the enterotoxin of Salmonella, which is a virulence factor that causes diarrhea.Interestingly, the stn gene has been shown to be present in all Salmonella spp.Regardless of their serotypes, this gene is used to identify Salmonella.Stn has toxic activity so it is a factor of salmonella virulence factors and is responsible for salmonella enterotoxigenic toxicity 9 .Plasmid-encoded fimbriae (Pef) play an important role in bacterial pathogenesis because they enhance the attachment of bacteria to the surface of the small intestine.These fimbriae are encoded by pef operon present in the virulence plasmid.The expression of pef genes is regulated by DNA methylation.Plasmidencoded fimbriae bind to antigens of the Lewis system that are predominant on the surface of human erythrocytes, skin epithelium and mucosal surface 10 .Salmonella possesses another type of fimbriae called Type 1 fimbriae, which are encoded by the fim operon and are assembled by the Chaperone-usher system.Type 1 fimbriae (T1F) are one of the most common organelles in the Enterobacteriaceae, including Salmonella species, and are important virulence factors, helping bacteria to adhere to the host cell surface 11 .Therefore, this study aims to diagnose salmonella by determining the sequencing of 16srRNA gene and determining some of its virulence factors.

Materials and Methods: Isolation and Identification
This study was conducted during the period from March 2019 to March 2020 in Tikrit city / Iraq.Salmonella was isolated from 350 stool samples taken from children under five years in age, suffering from diarrhea by using selenite broth and XLD agar.The developing colonies on XLD medium with a red color and black center were isolated.The isolates were identified to genus level by using a Vitek 2 compact system, in addition the isolates were diagnosed to a subspecies level by sequencing the 16srRNA gene.Extraction of Genomic DNA In this study, the Genomic DNA Purification Kit, manufactured by Promega in the United States of America was used to extract DNA.

Primers Preparation
The DNA primers mentioned in Table 1 were manufactured by Alpha Company in Canada as a lyophilized product.All the primers were centrifuged for a few seconds before use, after which the primers were dissolved in sterile deionized water to obtain the final concentration of each primer of 100 μM (stock solution), 10 μl of the stock solution were added to 90 μl of sterile deionized water to obtain the working solution of the primer at a concentration of 10 μM.

PCR Reaction
All PCR reactions were performed at volumes of 25μl in Eppendorf tube.A Laminar flow cabinet was used to prepare the reaction mixture.All reaction components were frozen separately and used at optimum concentration.Table 2 shows the components of the PCR reactions.The PCR reaction for the genes used in this study was performed according to the reaction conditions as in shown in

Electrophoresis
PCR products were electrophoresis on 2% agarose gel containing red safe stain at a concentration of 5 μl/100 ml of agarose gel.The wells of agarose gel were filled with 5 μl for each well, then the electrophoresis was carried out in two stages.In the first stage, a voltage differences of 2 volts/cm were used for 10 minutes, while in the second stage, a voltage differences of 5 volts/cm were used for 120 minutes.

Results and Discussion: Isolation
In this study 38 isolates were isolated from the 350 stool samples of children suffering from diarrhea, which are believed to belong to the Salmonella genus.Bacterial isolates were determined on XLD agar medium as in Fig. 1, according to the phenotypic shape, as rounded colonies that are 1-2 mm in diameter, red in color and have a black center in the middle, were selected 16 .

Figure 1. Salmonella enterica isolates Colonies on XLD agar Identification
The results showed that only 10 isolates belong to the genus Salmonella, the isolates were diagnosed to the level of the genus by using Vitek 2 compact system.The results of the molecular diagnosis using the 16srRNA primer according to the reaction conditions, mentioned in Tab.3 showed that all salmonella isolates possess 16srRNA gene.This gene appeared as a bundle on agarose gel with a size of 660 bp as in Fig. 2.Moreover, the sequencing results of 16srRNA gene of all Salmonella isolates showed a    --------------- ----------------

Detection of Virulence Genes
The virulence genes were detected by using the polymerase chain reaction according to the reaction conditions mentioned in Table 3

Detection of invA gene
The invA gene is an essential gene that is required for the invasion of epithelial cells of the intestines by Salmonella 17 .The invA gene in Salmonella contains a unique sequence of this genus, so this gene is also used in the molecular diagnosis of Salmonella 18 .The invA gene was amplified using the specialized primer as shown in Table 1 The results of electrophoresis on agarose gel of the PCR products showed that this gene appeared in all Salmonella isolates, with an appearance rate of 100% as shown in Fig. 4 The invA gene appeared as a bundle on agarose gel with a size of 284 bp as in Fig. 5.The results of the current study agreed with the results of Abd El-Tawab and his group 19 , as their results showed that the invA gene diagnosed in all Salmonella isolates was 284 bp in size.Also, the results of the study conducted by Al-Kaaby et al. 17 agreed with the results of the current study, as their results showed that all salmonella isolates possess the invA gene.The results of the current study also agreed with the results of the study conducted by Hussain et al 20 in Nasiriyah, as their results showed that the percentage of appearance of this gene was 100%.The results of the current study also agreed with the results of the study conducted by El-Sebay et al 21

Detection of stn gene
The results of electrophoresis on agarose gel of PCR products showed the presence of the stn gene in all salmonella isolates with a percentage of 100%, as in Fig. 4.This gene appeared as a bundle on agarose gel with a size of 617 bp as in Fig. 6.The results of the current study agreed with the results of the study conducted by Muthu et al. 22 , as the results of their study showed that all Salmonella isolates contain stn gene with an appearance rate of 100%.The results of the current study did not agree with the results of the study conducted by Ammar et al. 23 , as they stated that the appearance ratio of this gene was 58.82% of the total number of Salmonella isolates.

Detection of lpfA gene
The lpfA gene was amplified using the specialized primer lpfA as shown in Table 1.The results of electrophoresis on agarose gel of the PCR products showed the presence of the lpfA gene in all isolates of Salmonella, with an appearance rate of 100% as shown in Fig. 4.This gene appeared as a bundle on agarose gel with a size of 250 bp as in Fig. 7 The results of the current study agreed with the results of Webber et al. 24 , as the results of the study showed that all salmonella contain the lpfA gene with an appearance rate of 100%.Long polar fimbriae enhance the adhesion of salmonella to the M cells found in Peyer's patches in the intestine, which increases the virulence of salmonella, because these cells are the preferred entry site for salmonella.The results of the study also agreed with the results of Melo et al 25 as the results of their study showed that all salmonella isolates contain the lpfA gene with an appearance rate of 100%.

Detection of pefA gene
The pefA gene was amplified by using the specialized primer pefA as shown in Table 1.The results of electrophoresis on agarose gel of PCR products showed the presence of the pefA gene in 8 isolates, with an appearance rate of 80%.This gene appeared as a bundle on an agarose gel with a size of 700 bp as shown in Fig. 8.The results of the current study agreed with the results of the study conducted by Abd El-Tawab et al. 19 , as the results of the study showed that the appearance ratio of the pefA gene in the salmonella was 80%.The results of this study approximately agreed with the results of the study conducted by Shrivastav and his group 26 , as they stated in their results that the appearance ratio of this gene was 75%.

Detection of fimH gene
The fimH gene was amplified by using the specialized primer fimH as shown in Table 1.The results of electrophoresis on an agarose gel of the products of the polymerase chain reaction showed the presence of this gene in 6 isolates, with an appearance rate of 60%.This gene appeared as a bundle on an agarose gel with a size of 1008 bp as shown in Fig. 9.The results of the current study almost agreed with the results of the study conducted by El-Prince et al. 21, as they stated in their results that the rate of appearance of this gene in Salmonella isolates was 50%.The results of this study also differed from with the results of the study conducted by Saad et al. 22 in Egypt, as they stated in their results that the appearance rate of this gene was 100%.The results of the appearance of this gene also differed with the results of the study conducted by Ibrahim et al. 23 as they mentioned in their results that the appearance rate of this gene in isolates of salmonella was 100%.
-100% with standard gene sequence of Salmonella enterica subsp.enterica as in Table4.

Figure 2 .
Figure 2. Electrophoresis of PCR products of 16srRNA gene on agarose gel with concentration 2% at 70 volts/cm for 50 min.,The bands in the lanes 1-10 at a size of 660 bp representing the 16srRNA gene of Salmonella enterica subsp.enterica isolates.M: DNA ladder (100-1500bp)

Figure 3 .
Figure 3. Phylogenetic tree of the ten Iraqi isolates of Salmonella enterica subsp.enterica

Figure 4 .Figure 5 .
Figure 4. Appearance ratio of virulence genes in isolates of Salmonella enterica subsp.Enterica

Figure 6 .
Figure 6.Electrophoresis of PCR products of stn gene on agarose gel with concentration 2% at 70 volt/cm for 50 min.,The bands in the lanes 1-10 at a size of 617 bp representing the stn gene of Salmonella enterica subsp.enterica isolates.M: DNA ladder (100-1500bp)

Figure 7 .
Figure 7. Electrophoresis of PCR products of lpfA gene on agarose gel with concentration 2% at 70 volt/cm for 50 min.,The bands in the lanes 1-10 at a size of 250 bp representing the lpfA gene of Salmonella enterica subsp.enterica isolates.M: DNA ladder (100-1500bp)