The Three clinical isolates S. dysenteriae were collected previously from stool samples of patients suffering from severe diarrhea. The bacterial identification was re-confirmed by API 20 NE and API 20 E systems.  The report by Sethabutr and his co-workers mentioned that enteroinvasive E. coli (EIEC) is very closely related with Shigella species and has a similar pathogenic mechanism. Therefore, it is evident that many enteroinvasive E. coli could be called Shigella and vice versa, this relatedness makes the differentiation between them difficult and need serotyping for complete identification (Sethabutr et al., 1993; Grimont et al., 2007).  For the above reasons, only three clinical isolates were diagnosed correctly as S. dysenteria, after the initial diagnosis of many stool samples which was EIEC E.coli in all the other cases. The reconfirmation and differentiation between these isolates were done in Central Public Health Laboratory in Baghdad. This result indicates that the number of infections with S. dysenteriae is low despite the similarity of clinical signs and symptoms with E. coli. The asymptomatic carrier of S. dysenteriae is believed has an important role in sustaining the organism and in the spread of the disease within susceptible people. Our results were similar to Al-Musawi’s research team results that indicated the bacterial contamination with Shigella spp. was 3.2% (Al-Musawi et al.,2016) on the other hand, during military operation in Iraq, Shigella spp was (20%) isolated from US Marines stool samples in14 different battalion (Thornton et al., 2005). The survey research in both Bangladesh and Nigeria showed that the carrier rate of Shigella spp in children was 2–3% (Ghosh et al., 2014). However, reports from different Asian countries showed that incidence rates of infection raised by Shigella ranging 3 – 13% (Dhodapkar et al., 2008). The diarrheic stool infection that caused by S. dysenteriae may attributed to ability of this pathogen to adhere on the intestinal surface and induce diarrhea with pain , but the mortality is low or may cause by secretion  such as neurotoxin or enterotoxin from  S. dysenteriae (Richardson et al., 1992). In spite of the most pathogenic bacteria can produce different proteases and have the potential to breakdown the functional and structural proteins that comprise host tissues as well as to degradation important proteins in host defense. but in our study the ability of S. dysentria protease production was weak and small clear zone around the colonies occured with 1-1.4 mm diameter (table 1), the protease of Shigella classified into serine protease autotransporters (Sat), it is also, produced by diarrheagenic E. coli, it is enumerate among a significant virulence factors in both E. coli and Shigella (Henderson et al., 1998) which in turn is able to stimulate autoproteolysis of the mature protein from the Sat domain at the bacterial surface (Hendrixson et al., 1997; Stathopoulos et al., 2000)  also, the isolates  show low  hemolysis patterns on blood agar (table 1). But, the lysis of RBCs was observed in moderate intensity by hemagglutination test for non-heated bacterial supernatant in twofold dilution 1/10-1/320, while, agglutination appeared after treating the sample with heated supernatant which indicates that RBCs did not lyse (Table 2). The biofilm formation also was weak. The weakness in results may attributed to different reasons deal with involvement of many of the virulence factors under study or other factors such as growth conditions such as strain variation, origin, incubation temperature or, media constitute.

Table (1):
Hemolysis pattern and protease production of S. dysenteriae.

 Table (2):
Hemaaglutination test for heated and non-heated supernatant of S. dysenteries.

The agglutination appeared with heated supernatant treatment may attributed to the toxin was consider heat-labile and the lyses action was stopped by high temperature. This result can be concluded that hemolytic factor consider among virulence factors of S. dysenteriae because it lyse red blood cells .Using calcium ions to forming the transmembrane pores in the lipid bilayers of erythrocytes lead to loss of vitality and finely death of cells (Beutin et al.,1994). Some authors were reported that hemolysis was occurred by formation of a 25-Å pore within RBCs membrane not by hemolysis enzyme action but to action some proteins of type III secretion system like IpaB and IpaC insertion into RBC membrane. Ipa protein secretion and hemolysis were kinetically coupled processes during RBC-bacterial contact (Blocker et al., 1999).

The results of antibiogram profile were appearing in Table 3.  Results of many previous studies for antibiotic susceptibility testing were different; some of antibiotics were combatable and other incompatible with our result. Till now, we do not have a decent explanation for these variation results. S. dysenteriae has a variable behavior against different antibiotics. It was sensitive to ceftriaxone, ciprofloxacin and co-trimoxazole respectively; while resistant to tetracycline, chloramphenicol and ampicillin respectively , but Nalidixic acid was appeared moderate activity against  S. dysenteriae comparatively with control E. coli ATCC 25922.

Table (3):
The antibiotics susceptibility for S. dysenteriae.

Our results are compatible with the guidelines developed by WHO periodically, it was announced ciprofloxacin is drug of choice for infection by Shigella species (WHO, 2005).  According to local previous studies for antibiogram analysis by disc diffusion method, the clinical isolates of Shigella were isolated from patients whom suffering from diarrhea were resistant to Cefotaxime, Ampicillin, Streptomycin, Nalidixic acid, and Kanamycin (Suhad, 2007) whereas, Asaad K.T. and Afaf A. were noticed resistant to Rifampicin, Ampicillin, and Amoxicillin, but it appeared sensitive to Ciprofloxacin, Nalidixic acid, Neomycin and Cefotaxime (Asaad and Afaf, 2010). The antimicrobial drug profile in Switzerland European country during period 2004–2014 for S. dysenteriae were appeared resistance to Amoxicillin/clavulanic acid  Cephalothin,  Cefotaxime, Ciprofloxacin, Azithromycin, Kanamycin, Gentamicin and Nalidixic acid, but it were shown sensitive to Ampicllin, Trimethoprim, Sulfamethoxazole, Streptomycin, Tetracycline  and Chloramphenicol  (Nüesch-Inderbinen et al., 2016). The antibiotic susceptibility tests clarify that S. dysenteriae has a wide range of resistance to many antibiotics. This may attributed to the presence of the extra outer cytoplasmic membrane which comprised of lipoproteins, lipid bilayer, lipopolysaccharides and polysaccharides. Furthermore, misuse or abuse of natural and synthetic antimicrobial agents could be part of the involvement factors of resistance to antibiotics (Mordi and Momoh, 2009). In both medical and veterinary therapy, the source and period of microbial isolation, natural and the routine use of antibiotics may give rise to in wide prevalence of antibiotic resistance and development of antibiotic resistance genes particularly within the gram negative organisms. There are different mechanisms of antibiotic resistance include: decreased penetration of drug into the cell, efflux of antibiotics out the cell by efflux pumps, hydrolysis of antibiotics, and modification in target by mutation (Blair et al.,2015). The bla-TEM or bla-oxa genes are responsible for the development of ampicillin resistance, while resistance to chloramphenicol, streptomycin and tetracycline has mainly been attributed to the presence of catA1, strA and tetB genes respectively, but, the resistance to trimethoprim is associated with dhfrIa or dhfrIIIc genes (Toro et al., 2005).The first drugs used to treat Shigella infections were sulphonamides and followed by tetracycline and then by chloramphenicol. Shigella spp developed their resistance to all of these drugs and finely treatment was shifted to ampicillin and co-trimoxazole. Today, ceftriaxone, azithromycin and pivmecillinam are recommended by WHO against infection caused by fluoroquinolones- resistant Shigella species (Taneja and Mewara, 2016). From all above variations in susceptibility, the antimicrobial agents are of great value for bequeathing curative measures against microbial infections. Whereas, gradually development in resistance to these agents is a significant cause of attention and periodic monitoring of drug resistance of these organisms should be carried out in different geographical areas so that appropriate agent can be chosen for empiric therapy. Both mammalian RD and L20B cell lines were exposure to heat and non-heated bacterial supernatant of S. dysenteriae respectively. The oscillatory manner of the cytotoxicity analysis was appeared within 18 hr. and the pH value was changed within less than 24 hr. after treatment with non- heated and heated of filtrated supernatant.  Cell shape was shown changes include: seizure, shrinkage, segregation, rounding was observed as cytopathic effect. Cell death completely and lyses was notes with total destruction and caused either failure of cells in suspension to attach or detachment of cells from established monolayers within 120 hr. compared with non- treated cells as a control (Fig. 1). The pathogenesis of shigellosis or bacillary dysentery involve colonization, invade the epithelial cell in the lining of the colon, causing severe inflammation, due to Shigella cells grow and multiply intracellularly and spread to neighboring epithelial cells;  after that ulceration , hemorrhage and tissue demolition were occurred in last stage of shigellosis is the death of the cells that lining the colon and fibrosis of the colonic mucosa region, all these symptoms associated with abdominal pain and this sore results in the severe bloody diarrhea and may contain mucus, or pus. For this, Shigella cells may cause dysentery (Kodati et al., 2008; Hill Gaston, 2003). The abundance of extracellular secretion by Shigella spp has led to difficulty in characterizing these factors and to contravention in the enteropathogenicity.  In general, cytotoxic the cell may produce severe illness in humans. These morphological changes can defined as cytopathic effects (CPE) and can be useful to classify cytotoxic, this damage may attributed together some factors such as
i) Extracellular temperature – stable serine protease autotransporters (Sat) production to degrade components of extracellular matrix including collagens, laminin, elastin, and fibronectin, and finely cause the death of the cell after 120 hr. (Seshadri et al., 2006)
ii) Ipa (IpaB and IpaC) protein secretion that insertion into RD and L20B cells membrane (Schiavolin et al., 2013) and
iii) Shiga Toxin (an A-B toxin). Shiga Toxin causes cell death by preventing protein synthesis by cleaving a specific adenine residue from the 28s rRNA in the 60s subunit (Richardson, 1992). These explanations are compatible with the results of cytotoxic effect on RD and L20B caused by supernatant of Aeromonas hydrophila (Al-Rubai et al., 2011).

There is no available studies refer to ability of S. dysentria to cytotoxic of RD or L20B cell lines, but there are many articles showed the cytotoxic effect of  Shigella spp on other mammalian cell line.  In vitro this organism has competence to infecting monolayers of Henle intestinal epithelial cells and HeLa cells line (Hale et al., 1979) S. flexneri able to invade HeLa cells with high worthiness, This critical step in the pathogenic process is encoded by a 140 megadalton plasmid which induces phagocytosis of the bacteria by host cells. The virulent plasmid-associated hemolysin is a remarkable factor in the invasion and amplification of Shigella spp. in different mammalian cells (Sansonetti et al., 1986).The report by Brown and his colleagues explain that Shiga toxin produced by S. dysenteriae leads to inhibited protein and DNA syntheses in intact HeLa cells (Brown et al., 1980). Also, Shigella is able to induce necrosis and apoptosis of host cells, the infected cells by Shigella spp or other dangerous pathogen bacteria rapidly lose membrane integrity due to the release of effector proteins such as cytoplasmic lactate dehydrogenase enzyme through the type III secretion system and the exposure of phosphatidylserine. The Interjection of a pore by Shigella spp into the infected host cell membrane result the necrosis (Nonaka et al., 2003; Hersh et al., 1999). Chen and his colleagues reported that releasing IpaB by Shigella lead to kill the macrophage in infected host. This factor directly binds to and activates caspase-1 to become as the key molecule in the induction of caspase-1-dependent apoptosis by Shigella infection (Chen et al., 1996).  In the growth of the variety of virulent strain of bacteria have been appeared ability to produce spherical Nan vesicles called outer membrane vesicles (OMVs), these vesical carry many toxins such as heat-labile toxins and other virulence factors include lipopolysaccharides (LPS), DNA or RNA, lipids, and proteins (Lee et al., 2008;  Kesty et al., 2004).

These vesicles contain the same above factors that are known and contain other toxin such as Shiga toxin in OMVs of E. coli O157:H7 (Kolling .and Matthews, 1999), the cytolethal distending toxin in Campylobacter jejun vesicles (Lindmark et al., 2009) and the Cif protein in OMVs of Pseudomonas aeruginosa (Bomberger et al.,2009). In our study can conclude that the haemolytic pattern by IpaB and IpaC and Sat activity of the S. dysenteriae might play an important role in the pathogenesis of the organism in vivo thus occurrence of the cytotoxicity. Although, the results appeared some correlation between Sat activity and hemolysis with cytotoxic effects, may found other extracellular factors should be highlighted in relation to pathogenicity of S. dysenteriae. These factors might have diverse expression or under the same genetic control. It is not improbable that different virulence factors are serious under different event. These results clarify the presence of wide spread, multiple antibiotic resistant and cytotoxic S. dysenteriae. The linked between the hemolysis; proteolysis and cytotoxic activities must be further estimated to genetic control of other virulence factors, mechanisms genetically and network of these virulence factors in showed the pathogenesis of S. dysenteriae. ii) The destruction and damage of Both RD and L20B cell lines in our results by extracellular secretion produced by S. dysenteriae may effect on cancer cells and become weaker, the cytotoxicity of cancer cells are left alone vulnerable to attack by the active immune system and destroyed rapidly.  Although the toxins of S. dysenteriae are dangerous, they can be employed to treat cancer cells as an alternative therapy or in the development of anti-tumor drugs through destruction of tumor mass.