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Molecular Analysis of Shigella sonnei Strains Isolated from Hospitals of Tehran and Ilam Cities Using Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR)

Background and Objective : Shigella species belong to the family Enterobacteriaceae, which cause dysentery with abdominal pain and tenesmus in human. Our country is one of the endemic and occasional epidemic areas of the disease. In the present study, Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR) technique that has high differentiation and specificity power compared to phenotypic markers, was used to investigate diversity and geographical distribution of colones as well as typing of Shigella strains. Methods : In this study, a total of 40 Shigella sonnei samples were isolated from stool of patients with diarrhea and divided into 5 groups. After identification and confirmation of the isolates by biochemical and serotyping methods, one colony of each isolate was cultured on LB medium and after DNA extraction, PCR was performed. After electrophoresis of the PCR product, gel images were saved in the computer for further analysis and typing of the isolates. Result : In this research, 40 isolates were examined using REP-PCR and a similarity matrix was constructed based on Dice's coefficient. According to this matrix, some isolates were completely similar (genetic similarity coefficient= 1) and some isolates showed the least similarity (genetic similarity coefficient= 0). The dendrogram was obtained using the UPGMA algorithm. The calculated Cophenetic correlation coefficient for this dendrogram was 0.91645. Conclusion : From the results of the present study, it was concluded that we can type the Shigella sonnei strains using palindromic repetitive sequences. The extent of polymorphism indicates that REP-PCR technique is a useful method for genetic variation analysis in molecular typing of shigella sonnei strains. Although, some strains were completely similar, a high genetic variation was found among the studied population of Shigella sonnei . This level of variation is probably due to wide geographic distribution of this species in Iran.


Introduction
Shigella is a non-motile, non-spore-forming, capsuleless, Gram-negative, and facultatively anaerobic coccobacillus, which is 2-3 µm in size. This bacterium belongs to the family Enterobacteriaceae and humans are the only Our country is one of the endemic areas of the disease, wherein occasional epidemic outbreaks occur (2). In other countries, some researches have been conducted on recording molecular types of endemic and epidemic strains in order to trace the origin of the infection, but given the limited studies in Iran, no epidemiological relationship was found among the isolates (3).
Phenotypic and genotypic techniques are used to design effective strategies for the control of infections caused by this bacterium. Phenotypic techniques include evaluation of biochemical characteristics, phage typing, bacteriocin typing, immunoblotting, protein analysis, and identification of antimicrobial resistance patterns (4). These techniques are not powerful enough for identification and differentiation of S. sonnei strains and cannot determine the source and ways of distribution, and effective measures for controlling the spread of infection and prevalent isolates (5).
In recent years, genotypic techniques by specific molecular markers are used for epidemiological studies and typing of shigella species. These techniques include ribotyping, plasmid analysis, PFGE, chromosomal RFLP, RAPD-PCR, AP-PCR, REP-PCR, and ERIC-PCR (6-8). Among these techniques, REP-PCR has high validity and appropriate power for differentiation of isolates (9). Repetitive sequences of double-stranded DNA, which are called symmetric elements, are often seen in noncoding sections of DNA, (10).
Considering the presence of these repetitive extragenic palindromic sequences, by designing an appropriate primer according to desired sequence using PCR, electrophoresis of the resultant PCR products, and gel analysis by computer (6-9) and given the number of sequences in each strain and their difference in length, the number of bonds obtained by PCR for each strain is different, and therefore the strains are classified based on diversity of bonds. Although, use of PFGE is considered as a gold standard method, the REP-PCR is a simpler and lower-cost method and provides more rapid results (11). Considering that in our country, molecular studies on molecular epidemiology of shigella species are limited to specific regions, this research was carried out with the purpose of investigating genetic variation of S. sonnei strains using REP-PCR method (10).

Isolation and cultivation
In the present study, a total of 40 S. sonnei isolates isolated from patients with diarrhea, were analyzed. Then, the isolates were cultured on selective media, such as MacConkey agar and deoxycholate agar and confirmed by biochemical tests. A specific antiserum was used to differentiate the S. sonnei from other species.

DNA extraction
In order to extract DNA, first, one colony of the bacterium in LB media was incubated at 37°C for 18-24 hours. After incubation, the bacterial suspension was centrifuged at 3000 rpm for 5 min and the sediment was used for DNA extraction. DNA of the samples was extracted using Diatom kit, which has been designed based on GuSCN-Silica Gel method.

PCR
After DNA extraction, each sample was stored at -20°C.
The primer sequences used in PCR reaction were as follows: PCR was performed in a thermocycler with initial denaturation of 95°C for 5 min, then 40 cycles in 90°C for 30 s, 40°C for 1 min, 65°C for 8 min, and finally 75°C for 15 min for amplification of incomplete segments.

Observation of PCR products
About 10 µl of each PCR product was transferred to each well of the gel and electrophoresis was carried out for 1 h at 90 V. The electrophoresis gel was placed on a UV transilluminator and image was captured from the observed bonds. The gel images were stored in computer for further analysis and typing of the isolates.

Discussion
Seventeen scoreable fragments were produced using a pair of specific primer. Fig. 2 illustrates an electrophoresis pattern produced by PCR using REP-PCR primers. This level of polymorphism shows that REP-PCR technique is a useful method for analysis of genetic variation in molecular typing of S. sonnei strains. Estimates of genetic similarity based on this method revealed that some strains has 100% genetic similarity and there was no genetic similarity between the strain 23 and other strains. Estimation of genetic similarity using DNA fingerprinting data is a useful method for assessment of diversity and distribution of the isolates and strains involved in outbreaks and determination of the epidemiological relationship among different S. sonnei isolates.
In a study by Jersek et al. (1999), 64 isolates of Listeria monocytogenes isolated from human, animals, and food, were typed and classified into 4 genetic groups of A, B, B, and D (12) Snelling et al. (1996) investigated acinetobacter baumannii strains by REP-PCR typing and cloning boiling method and their results indicated that typing by this method is rapid and convenient (13). Beyer et al. (1998) used REP-PCR method to identify epidemic and non-epidemic isolates of Salmonella enterica that had source variation. Most of the epidemic isolates were assigned to groups 1 and 2, and non-epidemic strains were assigned to group 3 (14). In this study, it was concluded that S. sonnei strains could be typed using repetitive palindromic sequences.