Assessment of stn, sipB and sopB virulence genes in various Salmonella serovars

1. Introduction

Salmonella is a common cause of foodborne infections and is considered a major health problem worldwide ( 1 ). Based on reports published by the CDC in the United States, 40,000 cases of salmonellosis occur annually. Moreover, nontyphi salmonellosis has been reported to be the second most frequent type of foodborne infection in Europe. Salmonella infections are more common in countries with low sanitation standards with regards to the preparation and transportation of food and sewage disposal. Foodborne salmonellosis commonly occurs due to the consumption of meat products, fruits, and vegetables ( 2 ). Although more than 250 Salmonella serovars have been identified, infections in humans are caused by a limited number of serovars. Salmonella typhimurium and Salmonella enteritidis are two of the most frequently encountered serovars. Various serovars have shown a high capability to colonize in various animal hosts. Many of these serovars have a wide variety of animal hosts, whereas some, especially those causing illnesses among humans, have a limited number of hosts. The factors involved in bacterial virulence and colonization in hosts are complicated, and some of these factors include bacterial genes, gene expression, the level of the host’s immunity, bacterial response to immune systems, the host’s environment, and interaction with intestinal microflora ( 3 , 4 ).

Salmonella’s virulence largely depends on its ability to invade its host and survive within the host’s cells ( 1 ). Several genes are involved in creating a disease in the host, most of which are located in specific parts called pathogenicity islands in the bacterial chromosome ( 5 ). These pathogenicity islands are the result of bacterial evolution, and there are five major islands that play a role in virulence ( 6 ). The sopB gene is situated on pathogenicity islands 5, also known as SPI5 ( 7 ). This gene encodes a 62 kDa protein, which induces the rearrangement of the actin cytoskeleton and membrane ruffling ( 7 , 8 ). Additionally, it aids in the internalization of bacteria ( 8 ). The Salmonella pathogenicity island 1 (SPI1) encodes a set of invasion effectors, among which the Sips (Salmonella invasion proteins A-D) can be found ( 9 ). In the process of bacterial invasion, SipB protein plays a crucial role ( 10 ). Its remarkable resemblance to IpaB highly indicates potential to trigger apoptosis ( 10 ). As a result, this protein is considered a strong contender for inducing the process of programmed cell death ( 10 ). The stn gene of Salmonella plays a significant role in the organism's virulence ( 11 ). As with other enterotoxins that cause an increase in cAMP levels, stn stimulates a similar response ( 12 ). This response leads to an increase in the synthesis and release of prostaglandins, which may also contribute to the organism's enterotoxic response ( 12 ). In addition to the SPI genes, virulence plasmids, adhesins, flagella, and proteins linked to biofilm have important roles in pathogenicity and bacterial survival in host cells ( 13 ).

Considering the zoonotic nature of infection, the danger posed to human health and financial burden placed on health systems around the world to combat infections, prompt diagnosis and treatment is of the utmost importance. Furthermore, diagnostic methods based on bacterial culture and extraction are time- consuming; therefore, developing methods to detect Salmonella in a rapid manner is a crucial part of disease prevention, particularly during epidemics. Nowadays, molecular methods are considered the most rapid and sensitive means of diagnosis, and PCR is one of the most common among them. Bearing in mind the significance of certain genes in bacterial virulence, identifying them could be considered an important step towards understanding the pathogenic process, and, subsequently, developing better means to control and prevent disease. In this particular study, the abundance of three genes, specifically stn, sipB, and sopB in 103 Salmonella serovars isolated from livestock, poultry and humans was analysed using PCR and Multiplex PCR.

2. Materials and Methods

2.1. Culture and collection of serovars

In this study, 103 Salmonella serovars were analysed at the Microbiology Department of the Razi Serum and Vaccine Institute in Karaj. They consisted of 30 serovars from livestock, 30 from poultry, and 43 from humans (lyophilized or freeze-dried at -70°C). To culture the lyophilized samples, 10% horse serum was initially added to the TSB culture medium, and 2 ml of the mixture were added to the vials containing bacteria. The obtained suspension was cultured on blood, nutrient, and MacConkey agars. The plates were then placed inside an incubator at a temperature of 37°C for 24 h. The samples that had been freeze-dried at -70°C were cultured in the mentioned media and incubated at 37°C. Biochemical tests, including IMViC, TSI, catalase, oxidase, urease, and lysin were used to confirm Salmonella.

2.2. Serotyping of the samples

Salmonella serovars were identified based on H, O, and Vi antigens according to Kaufman-White guidelines. In this process, rapid slide agglutination with polyvalent antisera was used to confirm bacterial serology. Then, bacterial serogroups and serovars were determined using O and H antisera (phases 1 and 2), respectively.

2.3. DNA extraction

After performing confirmatory tests, bacterial precipitate was obtained by culturing bacteria overnight in nutrient agar, and was later used to extract DNA via boiling ( 14 ). Furthermore, after DNA was extracted, it was quantitatively assessed using spectrophotometry and qualitatively analysed by electrophoresis on gel agar.

2.4. Detection of virulence genes in serovars

Genotyping and identifying virulence genes stn, sipB, and sopB in the serovars under investigation was performed using PCR. In this process, 6 microliters of the master mix, one microliter of each of the forward and reverse primers (Table 1) with a concentration of 10 pmol and 2 microliters of template DNA with a concentration of 100 ng were combined, and double-distilled water was added to this combination to obtain a volume of 12 microliters. The PCR thermal cycles consisted of denaturation at 94 degrees, annealing temperatures according to the Tm of each primer, and expansion at 72 degrees centigrade. The end product of PCR was placed on agar gel, and the existence of specific bands in the serovars was assessed after electrophoresis.

2.5. Multiplex PCR

To carry out Multiplex PCR, 25 microliters of master mix, 1 microliter of each primer, and 3 microliters of template DNA were initially added to double-distilled water to reach a combined volume of 50 microliters. The Multiplex PCR protocol consisted of the following steps: (i) a host start step of 5 min at 94°C; (ii) 35 cycles, with 1 cycle consisting of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C; and (iii) a final extension step of 10 min at 72°C.). The final product of Multiplex PCR was placed on agar gel, and bands were assessed after electrophoresis.

3. Results

3.1. Biochemical results in Salmonella serovars

In this study, 103 serovars were analysed, consisting of 43 human serovars, 30 serovars from livestock, and 30 from poultry. The VP, indole, urease and lactase tests were negative in all samples, whereas the MR test was positive in all of them. Around 97.8% of serovars were lysin positive. Moreover, 89.32% of serovars changed the colour to blue in the citrate test, and 97.08% of serovars produced H2S in the TSI test.

3.2. Serotyping

Serotyping was conducted using slide agglutination with O and H antigens (phases 1 and 2); the serovars assessed are shown in Table 2.

3.3. PCR results regarding virulence genes

Based on PCR on the stn gene, the 617 bp band was observed in all 43(100 %) human serovars, 27(90%) out of 30 serovars from livestock and 29(96.6%) out of 30 serovars from poultry. In total, 96.11% of samples had this gene. The sopB gene, consisting of 220 bp, was detected in 43(100%) human serovars, 30(100%) serovars from poultry and 28(93.3%) out of 30 serovars from livestock. In total, 98.05% of the serovars possessed this gene. Regarding the sipB gene, which consisted of 875 bp, 43(100%) human serovars, 30(100%) serovars from poultry and 29(96.6%) out of 30 serovars from livestock possessed this gene. Overall, 99.02% of the serovars in the study had this particular gene (Figure 1).

Figure 1. Assessment of the presence of virulence genes stn, sopB, and sipB in the Salmonella serovars under investigation based on sources

3.4. Results of Multiplex PCR

To obtain more rapid results, multiplex PCR was performed. The results of Multiplex PCR on the products of electrophoresis are shown in Figure 2. As observed in the figure, sharp bands were observed for each of the three genes using this method.

Figure 2. Results obtained by electrophoresis of Multiplex PCR products for the virulence genes stn, sopB and sipB

4. Discussion

Salmonella is a prevalent food-borne pathogen that continues to pose a significant public health concern worldwide ( 15 ). The infection can manifest in human subjects as typhoid fever, gastroenteritis, bacteremia, and other associated extraintestinal complications ( 15 ). Given the presence of Salmonella in a variety of sources and its role as a zoonotic disease, timely diagnosis is crucial ( 16 ). Traditional serotyping techniques necessitate specialized knowledge and a range of antisera ( 17 ). Due to the time-consuming nature of traditional methods, such as culture and isolation, it has become necessary to use rapid techniques to identify the presence of Salmonella in suspected samples ( 16 ). This is essential for preventing the spread of the disease and potential epidemics ( 16 ). On the other hand, molecular techniques, such as PCR, afford a rapid, cost-effective, specific, and remarkably sensitive methodology, effectively overcoming their limitations ( 16 ). This study aimed to investigate the presence of three virulence genes, namely sopB, stn, and sipB, in multiple Salmonella serovars. In this study, the frequency of three genes (stn, sopB, and sipB) in 103 Salmonella serovars from humans, poultry, and livestock was assessed, and the results demonstrated that stn, sipB, and sopB could be detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively.

The stn gene is frequently detected in various strains of Salmonella and codes the enterotoxin protein. Other researchers have shown that this particular gene is preserved in various Salmonella serotypes ( 11 ). In a study conducted by Naik et al. to detect the virulent stn gene in goats and chickens in 2015, 32 isolates were identified, among which 100% possessed this particular gene. The results obtained in various studies showed that the stn gene is frequently detected in Salmonella isolates regardless of their source ( 18 ). In this study, 29 isolates out of 30 from poultry and 27 isolates out of 30 from livestock possessed stn, which was consistent with the results of previous studies. Another study conducted by Chaudhary et al. investigated the virulence genes in Salmonella serovars from pigs in 2015. Their results showed that from a total of 270 samples, Salmonella enteritides and typhimurium were detected in 37 samples. Furthermore, all 37 samples possessed stn, which signifies the importance of this specific gene in causing gastroenteritis in humans and also highlights the fact that it could be used to detect salmonellosis ( 19 ). Our results showed that 27 out of 30 serovars from livestock possessed stn and were consistent with the results of the mentioned study. Muthu et al. carried out a research to detect the stn gene in isolates of three different strains of Salmonella in humans in 2014. Overall, although this gene was present in 79.5% of the strains, the 2 typhimurium strains in their study lacked stn ( 20 ). Our study revealed that this gene was present in 100% of serovars from humans, regardless of the type of serovar; therefore, our results do not contradict their findings. Another investigation was performed by Makwana et al. with the objective of serotyping bacteria and detecting genes involved in virulence. A total of 284 samples of meat were assessed, and 13 Salmonella isolates were obtained, all of which possessed the stn gene ( 21 ). Overall, it appears that stn is an important factor in bacterial virulence, regardless of the source and serovars, and that it can commonly be detected.

The sopB gene codes a protein with inositol phosphate phosphatase activity, which is transported via a SPI-1 dependent pathway inside host cells ( 8 , 22 ). This specific protein causes the secretion of chloride ions and leads to diarrhea in the host ( 7 ). Osman et al. conducted a study to identify the integrons and gene cassettes responsible for drug resistance among multiple drug resistant serovars isolated from humans in Egypt in 2014. The SopB was detected in all the Salmonella isolates, which was similar to the findings of the present study ( 23 ). Elemfareji et al. carried out a study to identify virulence genes among Salmonella typhi and nontyphi strains in 2013, and the results demonstrated the presence of the sopB gene in all the Salmonella typhi and enteritidis strains, regardless of the source ( 24 ). In our study, the sopB gene was detected in 101 serovars, regardless of the source, which is consistent with their findings. Mezal et al. carried out an investigation to detect Salmonella enteritidis in poultry in 2014. In this study, 60 samples were assessed in terms of antibiotic resistance and virulence genes, and all isolated were found to possess sopB and sipB ( 25 ). In our study, 14 out of 15 samples of Salmonella enteritidis had sopB and sipB, which was consistent with the results obtained by this group of researchers. Rasha et al. conducted a study to investigate the virulence genes in nontyphi Salmonella strains in 2015. Their results showed the presence of sopB in 5 strains isolated from chickens and humans. Salmonella typhimurium also possessed the stn gene ( 26 ). In the present study, the results of PCR for sopB and stn genes in nontyphi isolates (in humans and poultry) showed that sopB was present in all serovars, and that only one of the serovars from poultry lacked stn, which was in contradiction with the results obtained by this particular group of researchers.

The sipB gene codes a protein responsible for the invasion of host cells. This specific protein is necessary for the transport of other proteins, and in addition to this role, it possesses effector activity in pathogenicity ( 10 ). Kim et al. performed a study to assess the molecular characteristics of drug resistant nontyphi Salmonella isolates from chickens in Korea in 2017 ( 27 ). Their results revealed the presence of sopB and sipB in all serovars isolated from poultry, which was consistent with our findings. Beshiru et al. conducted a study to investigate virulence genes in Salmonella serovars from shrimps in 2019, the results of which revealed the presence of sipB in 78.6% of isolates ( 28 ). In our study, 99.02% of all serovars possessed sipB, which was in contradiction to the findings of this group of researchers. Ed-Dra et al. carried-out research to assess antibiotic resistance and detect virulence genes in Salmonella strains. Their findings demonstrated that all isolates possessed sipB and were resistant to at least one antibiotic ( 29 ). In the present study, sipB was detected in 102 out of 103 serovars (regardless of the source); therefore, our findings were consistent with those reported by the mentioned group of investigators.

A study was conducted by Najafi et al. to identify cmlA/tetRbla_TEM, bla _PSE-1, and sipB in Salmonella strains in Iran in 2017. In this particular investigation, 163 clinical samples from patients who had been referred to hospital were collected, among which 48 cases of Salmonella were detected. Out of these 48 samples, 25 had Salmonella enteritidis, while 14 had Salmonella typhimurium, and 9 had Salmonella infantis. The M-PCR results demonstrated that 16.6% of isolates had sipB ( 30 ). In our study, all 43 isolates from humans possessed sipB, which was contradictory to the results obtained by this group of researchers.

In summary, the findings of this study revealed the presence of stn, sopB, and sipB in various Salmonella serovars. The frequency of these in different studies has been varied, which could have been due to different study regions, various sources or differences in terms of serovars. Having said that, one noteworthy fact is the high prevalence of these specific genes, and this could be taken into consideration when adopting policies to fight salmonellosis. Our study results revealed that stn, sopB, and sipB were present in all isolates from humans. Moreover, sopB and sipB were detected in all isolates from poultry, whereas stn was detected in 96.6% of these isolates. Among isolates from livestock, stn was present in 90%, while sopB and sipB were detected in 93.33% and 96.66%, respectively. Based on our findings, these genes can be detected in Salmonella isolates regardless of the source. All Salmonella typhi serovars (4 isolates), typhimurium serovars (10 isolates), paratyphi A serovars (8 isolates), paratyphi B serovars (8 isolates), and infantis serovars (10 isolates) in this investigation possessed stn, sopB, and sipB, which showed the presence of these genes in various isolates, regardless of serovars. Considering the significance of these genes in bacterial virulence, their presence in serovars isolated from different sources and the importance of detecting them in understanding pathogenicity and potentially preventing disease, they could be used for molecular techniques such as PCR.

Authors' Contribution

This article was the result of M. J. S.’s master’s thesis, and S. M. B. was the supervisor and P. K. was the advisor.

Ethics

The authors declare all ethical considerations were respected in the preparation of the submitted article.

Conflict of Interest

The authors declare that they have no conflict of interest.

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