SHigella flexneri is a low-dose infectious microorganism that swallowing a small number of it (about 100 bacteria/ml) can cause infection. This is the main reason for the high prevalence of the disease, which is estimated for 165 million infected people and 1.1 million deaths worldwide per year (1, 2). Similar to other diarrheal diseases, shigellosis is treated with intravenous serum injection. Shigella infections respond to the antibiotic treatment by reducing fever and diarrhea, but there have been several reports of the antibiotic resistance (3). Therefore, to deal with the infectious agents resistant to antibiotics several studies must be conducted on pathogenicity, ways to escape the immune system, and resistance to antibiotics. In this study, the biofilm formation of Shigella flexneri and its association with the changes in the expression of TA (Toxin-Antitoxin) genes were investigated (4, 5).
Biofilms are aggregates of microbial colonies that form a cellular matrix containing a protective polysaccharide layer. The biofilm formation is the major factor involved in bacterial survival and resistance (6). In general, bacterial resistance in biofilm condition is much higher than planktonic state; one of the main reasons for this resistance is the polysaccharide matrix, which prevents the antibiotics penetration. In addition, lower level of bacterial metabolic activity in biofilm is another reason for the resistance to antibiotics (7).
Studies on Shigella flexneri have shown that prolonged exposure of the bacterium to the bile salts leads to biofilm formation (8). In addition, salt concentration can significantly affect the capacity of Shigella to produce biofilm in microplate wells (9). Ferulic acid has also been reported to limit the formation of biofilms by Shigella flexneri (10). These indicate the effect of different salts and acids on the biofilm formation of this bacterium.
On the other hand, in the case of TA systems, recent studies have shown that under stress condition, antitoxin is selectively degraded, which causes toxin activation and function (11, 12). It has been suggested that toxins in these systems are associated with various cellular processes such as regulating gene expression (13).
Several evidences have shown that these systems are involved in the formation of biofilm and Quorum sensing (14, 15). Lon protease is a protected and ATP-dependent serine protease in bacteria that selectively degrades mutated and abnormal proteins as well as some short-lived regulatory proteins such as antitoxins. Thus, by releasing the toxin it is activated. This protease plays a pivotal role in different bacterial mechanisms, including cell differentiation, participation in biofilm production, and bacterial survival (16, 17).
Since there was not much information about the existence and prevalence of these systems in Shigella flexneri, and due to the regular updating of the databases related to these systems, we evaluated the expression of these genes in the cell culture following cell infection by Shigella flexneri during a study after the identification of TA systems by these databases as well as the design of specific sequences of the related primers with the help of authentic software. This study was conducted in line with our previous study (18). The aim of this study was to investigate the changes in gene expression compared to the control by examining the biofilm of Shigella flexneri.
Bacterial Preparation and DNA Extraction
This study was performed in 2019 in the laboratories of Iran University of Medical Sciences. The standard strain of Shigella flexneri (ATCC 12022) was prepared from the Microbial Bank of the Department of Microbiology, Iran University of Medical Sciences. The bacteria was cultured on Hektoen enteric agar (HEK) (Merck, Germany) and incubated at 37°C for 24 hr. The boiling method was used to extract genomic DNA (19). The quality and concentration of the extracted DNA were evaluated by Nanodrop (Nanodrop Technologies, Wilmington, De, USA). After reading the adsorption ratio of 260/280 and ensuring the purity of DNA, the sample was stored in a sterile microtube at -70°C for further use.
Polymerase chain Reaction and Electrophoresis
PCR was performed in a thermal cycler (Bio-Rad, USA) using Master Mix (Fermentas, Lithuania) (12.5 µL) and extracted DNA (1 µL). The specific forward and reverse primers were used at 10 pM concentration (1 µL/each) (18). The final volume reached to 25 µL using nuclease-free water. The cycling program was as follow: an initial denaturation step at 95°C for 5 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 25 sec, and a final elongation phase at 72°C for 5 min. The PCR products were electrophoresed on 2% agarose gel. For this purpose, 2 gr of agarose powder (Sigma-Aldrich) was dissolved in 100 ml of 0.5X TBE buffer. After gel preparation and adding DNA Safe Stain, it was transferred to the tank and the samples were loaded in the wells. Finally, the bands were examined and photographed using gel doc.
Quantitative Evaluation of Biofilm Formation by Microtiter Plate Method
Shigella flexneri was cultured in trypticase soy broth (TSB) medium containing 1% glucose for 24 h at 37°C, and then a suspension equivalent to half McFarland was prepared. Then, 200 µL was inoculated into the wells of 96-well microplate. After incubation for 24 h at 37°C, the wells were washed three times with PBS to remove unabsorbed bacteria. The absorbed bacteria were then fixed for 15 min using 95% ethanol and the plate was air dried. The wells were stained with 200 µL of 0.02% crystal violet for 5 min. The microplate was then washed twice with distilled water to remove the excess dye. After drying, 200 µL of 33% glacial acetic acid was added. Following the contents fixation with ethanol, the adsorption of the dye dissolved in acetic acid was measured at 492 nm wavelength. The culture medium alone was used as negative control. Thus, to quantitatively evaluate the ability of Shigella flexneri to produce biofilm, the average adsorption of three wells was calculated and compared with the average adsorption of three control wells (uncultivated medium). The ability of bacteria to produce biofilm was determined. The ability to form biofilm was obtained through Table 1. To ensure the accuracy of the work, these tests and measurements were repeated thrice at different times (20).
Table 1. Calculating the ability of biofilm formation compared to the control
Degree of biofilm production | Intensity of colorimetry |
Lack of production | OD< ODnc |
Weak | ODnc< OD< 2×ODnc |
Medium | 2×ODnc< OD< 4×ODnc |
Strong | 4×ODnc< OD |
Investigation of Gene Expression in Biofilm using qPCR and Livak Method
At 8 and 24 h after induction of biofilm formation in Shigella flexneri, total RNA was extracted using RNA extraction kit (Roche, Germany). To measure the concentration and purity of RNA, the amount of optical density was read using nanodrop. After ensuring the purity of the extracted RNA, cDNA was synthesized using cDNA synthesis kit (GeneAll Biotechnology, South Korea). The 16S rRNA gene was used for the data normalization. Then, the expression of the studied genes was measured in the presence of internal control gene compared to the control sample. All experiments were performed thrice.
Each sample was done in duplicate. For each gene, two microtubes without template were placed to ensure the absence of contamination. To prepare the reaction mix with a total volume of 20 µL, 5 µL of sybr green Master Mix (qPCR Master Mix, Bioneer, Korea), 1 µL of cDNA, specific forward and reverse primers with 10 pM concentration (0.5 µL/each) and 13 µL nuclease-free water was used. All the steps were performed on ice. After spinning for 10 sec, samples were placed inside Rotor Gene thermal cycler (Corbett, Australia). The thermal cycling consisted of an initial activation step at 95°C for 10 min, 45 cycle of denaturation at 95°C for 15 sec, annealing and extension at 60°C for 30 sec. Real-time PCR results were analyzed using Rest software and the amount of gene expression (fold change) was calculated using Livak method (18, 21).
Statistical Analysis
All data was presented based on three replications in each experiment and the mean of the measurements was performed in three replications and its statistical analysis was determined by analysis of variance (ANOVA) and P-value<0.01 using SPSS 22 (SPSS Inc., Chicago, Ill., USA).
Polymerase Chain Reaction Test Result
Results of Shigella flexneri Biofilm Formation and Genes Expression
The ability of the studied Shigella flexneri strain to produce biofilm was evaluated by microtiter plate method using ELISA reader. The results showed that the tested strain was able to produce biofilm strongly. The qPCR results showed a fold increase in the expression of TA systems and lonP genes at 8 and 24 h after biofilm formation compared to the control. The results of the expression of the studied genes in the mentioned hours are shown in Figures 2 and 3, respectively (P<0.01).Figure 2. Results of expression of the studied genes in biofilm conditions compared to the control sample at 8 hour (results normalized in the presence of 16S rRNA internal control gene) (P <0.01)
Figure 3. Results of expression of the studied genes in biofilm conditions compared to the control sample at 24 hour (results normalized in the presence of 16S rRNA internal control gene) (P <0.01)
According to the obtained results for the TA systems increased expression level in terms of biofilm formation, it is possible to understand the association between these systems and biofilm formation. Hemati et al., in 2014, using PCR method, showed that in the clinical isolates of Pseudomonas aeruginosa, which have a high ability to form biofilms, TA systems and Quorum sensing genes are abundant (22), which indicates the association of biofilm formation with the presence of TA systems. This is consistent with the results of our study. In a study in 2016, Wood et al., identified a type II TA system called HigB/HigA in Pseudomonas aeruginosa PA14 and showed that HigB toxin affects biofilm production and virulence factors, and thus this system affects the pathogenicity of this strain (23).
In 2017, Valadbeigi et al., investigated the effect of a compound called PNA on the expression of the mazE antitoxin gene and its effect on biofilm formation in 18 clinical isolates of Pseudomonas aeruginosa. They mentioned that mazE antitoxin gene could be targeted for controlling the biofilm production by Pseudomonas aeruginosa (24).
In 2018, Chan et al., identified four TA systems, including pezAT, yefM-yoeB, relBE, and phD-doc in Streptococcus pneumoniae. They showed that strains lacking the yefM-yoeB system, as well as mutants in both yefM-yoeB and relBE systems, had a significant reduction in biofilm formation ability (25). This indicates that, similar to our results, there is a significant association between the presence of some TA systems and biofilm formation.
In a study in 2019, Alhusseini et al., examined 54 isolates of Pseudomonas aeruginosa MDR and evaluated the presence of five type II TA systems, including relBE, hipBE, mazE, ccdAB, and mqsR. They observed biofilm formation in 90.74% of the isolates. Also, PCR results for identifying the gene loci of TA systems showed a very high percentage of these systems in the studied isolates (26). These results were similar to the present study outcome.
In a study by Ma et al., in 2019 conducted on 3 strains of Staphylococcus aureus, the role of the MazEF system in infections as well as the association between this system and increased biofilm growth was demonstrated (27).
Numerous results of other bacteria have shown that maz toxin helps bacteria survive in harsh conditions. This system was also present in our studied strain. In Myxococcus xanthus, a Gram-negative bacterium, MazF plays an important role. It is induced by the formation of a spore-producing body, resulting in the death of 80% of the cell population by cell lysis and the remaining 20% of the cells are capable of forming spore-producing bodies (28).
Kwan et al., in a study in 2015, examined the MqsR/MqsA type II system in Escherichia coli and showed that due to the high concentration of deoxycholate in the gallbladder and upper intestine, this system is physiologically important and vital for the growth and survival of Escherichia coli cells in these areas, which have a high concentration of bile salts (29).
Targeting the important toxins and their regulating protease is suggested as an appropriate method of treatment. One method to target a gene is antisense technology. Javanmard et al., in a study in 2020, designed and applied antisense against cagA gene in Helicobacter pylori and showed that antisense along with penetrating peptide could be used as an effective tool to inhibit the target gene mRNA (30).
In the present study, the expression of genes was evaluated in biofilm condition compared to the control with lack of biofilm formation. The results showed that GNAT and maz toxins genes expression had a significant increase in biofilm conditions compared to the control group. These results suggest that these two systems may contribute to the formation of biofilms. In addition, due to the increased expression of Lon protease gene, its importance and necessity in bacterial survival can be realized through regulation and control of TA systems in biofilm conditions. Therefore, this protein can be a favorable target for introducing new therapeutic compounds.
We would like to express our gratitude to all the people who cooperated effectively during this study. It is worth mentioning that this article is the result of a part of Mr. Erfan Kheradmand's doctoral dissertation from the Islamic Azad University, Science and Research Branch.
This article is an independent study that was conducted without organizational financial support.
Conflicts of Interest
The authors declared no conflict of interest.
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