Bacterial isolate identification
Four samples (Salmonella-positive samples) of the total 32 examined tahini samples were found to be infected with Salmonella. These isolated bacteria gave the specific characteristics of Salmonella: dark colonies with black center on XLD, black colonies on Hektoen enteric agar, negative urease reaction on urea agar, sulfite reduction on triple sugar iron agar (TSI) and lysine iron agar (LI) with gas formation in TSI, negative β-galactosidase, negative indole, and negative Voges-Proskauer test. The confirmatory tests using poly O-Vi (somatic and capsular) antisera and poly H (flagellar) antisera were performed, and the results showed that the isolated bacteria are Salmonella.
For molecular identification of the Salmonella isolates, the 16s RNA gene analysis was performed. The PCR fragment of 475 bp was amplified and sequenced. Then, sequence BLASTN analysis was performed on the GenBank (NCBI) databases. A 99% sequence similarity was found to be with Salmonella enterica subsp. enterica, which indicates that the isolated bacteria are Salmonella enterica subsp. enterica serotype choleraesuis. The sequence of 16s gene of the isolated bacteria was deposited in NCBI GenBank and was given the following accession no. MK041288.1.
Moreover, serological analysis was performed to determine the specific serotype as the 16s RNA analysis is not a precise method at the levels lower than species in bacteria. Therefore, full Salmonella serological identification was performed on three isolates from the four positive samples. The results of the full serological tests showed that the antigenic formula of the three bacterial isolates is somatic antigen 6, 7; flagellar antigen phase 1: c; and flagellar antigen phase 2: 1, 5. By referring to the Kaufmann-White-Le Minor scheme [22], the bacterial serotype was confirmed as Salmonella enterica subsp. enterica serovar choleraesuis.
For identification of Salmonella serotypes, till now, serotyping using antisera and pulsed-field gel electrophoresis (PFGE) is the only accredited traditional methods. However, serotyping using antisera has an advantage of being cheaper than any other DNA dependent methods. Even the PFGE method is still expensive and takes much time for comparing bacterial serotypes [25]. In addition, PFGE does not show a unified profile for different strains of the same serotype [26]. On the other hand, the rates of concordance at the species level by using partial sequencing 16s rRNA gene and sequence alignment technique were found to be 80% [27]. Therefore, it seems that using 16s rRNA analysis is not trusted at taxonomic levels lower than species. So, in this study, we considered the partial sequencing of 16s rRNA gene as a primary identification method and the results were confirmed using the serotyping procedure. Some new serotype identification techniques are depending on 16s rRNA like 16s rRNA PCR-high-resolution melt analysis assay (HRMA), which is quite accurate [28] however it still needs more data to cover all serotypes of Salmonella [29]. Therefore, about 2600 serotypes are reported because of limited resolution and lower sensitivity of 16s rRNA gene analysis compared to metagenomic sequencing method [30, 31].
Thermotolerance analysis
In order to evaluate the thermotolerance of the isolated bacterium (serotype), S. choleraesuis in parallel with Salmonella typhimurium ATCC 13311 (reference bacterium), D value and Z value determination of both serotypes in aqueous nutrient solutions at water activity of 0.98 was performed under different temperatures 60 °C, 65 °C, 70 °C, and 75 °C. The results showed that the D values of S. choleraesuis at the previously mentioned temperatures are 45.6, 33.6, 21.6, and 9.6 s, respectively, while the corresponding D values for S. typhimurium were 43.2, 35.4, 16.2, and 3.6 s, respectively. The Z values were found to be 4.9 and 4.5, respectively. Therefore, based on our results, thermotolerance in the isolated strain of S. choleraesuis is relatively more than in S. typhimurium (ATCC 13311). It looks like that there is a wide range of thermotolerance variability of Salmonella among different serotypes and even among different isolates of the same serotype. It was reported that the D values of Salmonella agona at the temperatures 60 °C, 65 °C, and 70 °C in buffered peptone solution were 148, 19.8, and 7.8 s, respectively. These values are considered very high D values for this serotype of Salmonella enterica, while the D values of Salmonella typhimurium at the same temperatures were 13.2, 6, and 1.2 s. It has been shown that the Z value of Salmonella spp. ranged between 3.9 and 7.4 °C [32] which is in accordance with the Z value obtained in our study. However, their D values are still higher and lower compared to our results.
It has been reported that the D values at 60 °C and 65 °C of Salmonella spp. in beef meat were 8.6 min and 1.5 min, respectively [4], while according to [34,35,36], the D values of Salmonella spp. at the same temperatures and in the same product were 5.3 and 0.53 min, respectively [33], indicating variability in the D values of Salmonella spp. The main cause of the high values in the previously mentioned study could be due to lower water activity. In addition, Liu et al. [4] showed that 70 °C is a critical temperature for Salmonella death, which is coherent with the results of the present study. Moreover, high thermotolerance of Salmonella in the aqueous solutions with high water activity was reported, where the heat treatment up to 85 °C for 1 min did not eliminate the naturally occurring contaminant from alfalfa seeds [34]. This finding is in parallel with our results indicating that Salmonella can endure high temperatures in aqueous solutions.
In our study, statistical analysis showed a significant difference between the D values of both bacteria S. choleraesuis and S. typhimurium under three temperatures 65 °C, 70 °C, and 75 °C (Fig. 1). Also, the thermotolerance superiority of S. choleraesuis on S. typhimurium was found. In addition, there was a significant difference between the obtained Z values of both serotypes (were determined depending on the obtained D values) 4.9 and 4.5 degree, respectively (Fig. 2). Growing both serotypes at the same conditions indicated that the difference in D and Z values is due to genetic factors and it is neither temporarily acquired nor due to the environmental conditions. These obtained results are consistent with the findings of Alvarez et al. [9], Dodier [35], and de Melo et al. [36], where it was reported that there is a difference in thermotolerance among S. senftenberg, S. typhimurium, and S. enteritidis serotypes.
Relative gene expression analysis
The expression of DnaK and HtrA genes in both serotypes was investigated to find out if there is a correlation between the expression of these genes and thermotolerance. Relative expression analysis using qRT-PCR was performed on the bacteria grown under different temperatures (37 °C, 42 °C, 47 °C, 50 °C, and 55 °C), while the bacteria grown at 37 °C were used as control. In our study, the relative gene expression of DnaK in S. choleraesuis at the different temperatures 37 °C (control), 42 °C, 47 °C, 50 °C, and 55 °C were 1.00, 44.32, 46.21, 47.50, and 39.67, respectively (Fig. 3). The highest relative gene expression of DnaK was at 50 °C, and then, its expression started to decline but not sharply. By contrast, the DnaK gene expression at the different temperatures in S. typhimurium were 1.0, 41.07, 47.18, 38.32, and 32.90, respectively. It is obvious that the highest DnaK expression in S. typhimurium was at 47 °C, and these results are in the same ranges of previous measurements [8]. On the other hand, the HtrA relative expression was found to be higher in S. choleraesuis than in S. typhimurium (Fig. 4). The relative HtrA expression was 1.00, 4.41, 5.10, 3.92, and 4.17, respectively, in S. choleraesuis, while it was 1.00, 4.26, 4.06, 3.71, and 3.78, respectively, in S. typhimurium; this range of gene expression levels is matching with the results of Baron et al. [37], where they reported that the expression fold of HtrA after induction increased up to 4.83–7.79 times. The highest HtrA expression was at 47 °C in S. choleraesuis, while it was at 42 °C in S. typhimurium. Therefore, it is clear that the peak of relative gene expression of both heat shock genes (DnaK and HtrA) comes more lately in S. choleraesuis serotype.
Our results of qRT-PCR analysis showed that the relative expression of two studied heat shock genes DnaK and HtrA increased with increasing temperature. These results are in accordance with the findings of Sirsat et al. [8], where they found that the expression of HtrA and DnaK were upregulated up to 4.11- and 44-fold, respectively, at temperatures over 40 °C. In addition, in this study, the gene expression of both genes DnaK and HtrA in both serotypes increased dramatically with increasing temperature. Moreover, when the temperature is shifted from 37 to 42 °C, there was a sudden increase in gene expression indicating a threshold in this range of temperature, which is also coherent with the previously mentioned study [8]. Also, a significant difference in the relative expression of HtrA gene between S. typhimurium and S. enteritidis was reported, which was accompanied with relatively higher thermotolerance of S. typhimurium that has the higher gene expression [38]. This result is also in accordance with our results.
Comparing the results of relative gene expression of DnaK and HtrA in both serotypes using Tukey’s multiple comparisons test indicated that the trend of relative expression of both genes is higher in S. choleraesuis than in S. typhimurium. Moreover, the difference of relative expression of both genes is significant and relative gene expression has a correlation with the bacterial thermotolerance. This result also meets the results of Yadav et al. [38] in their study on thermotolerance difference between S. typhimurium and S. enteritidis.