Profiling and Curing from Shigella Spp Isolated from Plasmid Diarrheal Patients

Problem statement: Shigellosis is a common infectious disease especia lly in underdeveloped countries. The bacteria are primaril y transmitted through the faecal-oral route. The inflammatory process of acute Shigella infection affects the colon and is characterized c linically by fever, cramping abdominal pain with frequent loose stools that might contain mucus, pus and blood. Approach: This study determined the susceptibility of Shigella isolates to antibiotics. Afterward, plasmid isolation from pathogenic Shigella will carried out to achieved information concernin g to the presence of plasmid DNA in Shigella isolates. Plasmid curing will be done to seek corre lation between resistance to antibiotics and plasmid occurrence in Sh gella. Results: We found that the incidence of diarrhea in male is almost similar to female. The d istribution of Shigella spp., in male more than in female represented 54.54 and 45.46% respectively. Conclusion: Approximately 89.5% of the diarrhea cases had no bacterial pathogen, suggested of proba bility of viral infection.


INTRODUCTION
Shigellosis is a common infectious disease especially in underdeveloped countries. WHO bulletin concluded that, 99% of the estimated 165 million cases of Shigella diarrhoea annually occurs in developing countries. Majority (69%) of episodes are seen in children under five years of age 3. This is attributable to personal hygiene and sanitary conditions which promote spread of organisms like Shigella and other enteric pathogens (Kotloff et al., 1999;Hawari, 2008).
Four serogroups (or species) of shigella have been described including group A (Shigella dysenteriae), group B (Shigella flexneri), group C (Shigella boydii) and group D (Shigella sonnei). These groups are further classified into serotypes and sub-serotypes. This serotyping scheme uses the polysaccharide O antigen found in the outer part of the cell wall. Shigella organisms are highly virulent. A very small inoculumas little as ten microorganisms-can cause disease in humans (Ozuah, 1998;. Antibiotic resistance among enteric pathogens is of great importance in the developing world, where the rate of diarrhoeal disease is high. Continued mismanaged selective pressure has contributed towards the emergence of multiple drug resistant bacteria and that has been regarded as an inevitable genetic response to antimicrobial therapy (Smith et al., 2003).Frequency of antibiotic resistance among Shigella species is growing up and has been reported in various studies globally.
The ability of a genetic marker for transferring from one bacterium to another through conjugation or transformation provides a good presumptive evidence for the involvement of plasmid, particularly if the transfer frequency is high. Moreover, loss of certain genetic markers as a result of treatment of bacterial cell to plasmid curing agents also suggests for the plasmidial nature of the marker (Mesas et al., 2004;Altalhi, 2007). The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance (Alhaj et al., 2008). Plasmid curring: The procedure of Zurkowski and Lorkiewicz (1978) was used for heat curing. Overnight YEM broth cultures were inoculated into PA broth. The cultures were incubated in 35, 37, 40 and 45°C water bathsand given daily 5 sec. blending in a Vortex mixer for aeration. The cultures were transferred to fresh broth at weekly intervals. After heat treatment, the cultures were plated on YEM agar and the plasmid profiles of single colony isolates were observed.

Curing of plasmids by ethidium bromide:
Techniques were similar as described by Ansari and Khatoon (1997). The plasmids were studied for curability in isolated colony. N.B. broth (5 mL) tubes containing graded concentration of Ethidium Bromide (EBr) were incubated with log phase cultures of Shigella isolates host bearing R plasmids to give a 20 fold dilution. A control tube lacking EBr was always included. All the tubes were incubated over night at 37°C. The contents of the control tube and of the EBrcontaining tubes were plated on MA, to obtain isolated colonies. After overnight incubation at 37°C, these were replicated on antibiotics containing plates to check for the loss (or its absence) of antibiotic resistance determines.
Curing of plasmids by sub culturing: Kamiunten (1990) method used with some modified as fallow, Shigella isolates had optimum growth temperature of 37°C a. Bacterial strains were grown in 5 mL of YP medium at 37°C for 24 h and then 0.1 mL of the cell suspension was transferred to 5 mL of the fresh same medium and shake-cultured at 37°C for 24 h. After twelve times of subculturing under the same conditions, the cultures were diluted with sterile distilled water and plated on YP agar medium. After 2 or 3 days of incubation at 37°C. These clones resulted from streaking on the medium were subcultured for examination of their plasmid content.
Plasmid DNA isolation: The alkaline lyses method (Kado and Liu, 1981) was used for plasmid DNA isolation.

RESALTS
In this study four hundred patient with acute diarrhea were involved. They were 232 males (58%) and 168 females (42%). The age ranged between months to 70 years as show in (Table 1).
Like many other developing countries, diarrheal diseases are among the main health problems in Iraq, During the study period, 400 stool samples were received were found to be positive for Shigella spp., 11 isolates (2.75% from total samples), 42 samples gave positive results, but the results were canceled and focusing on Shigella spp., (26.1% from total isolates). The frequency of isolation of Shigella spp., in our study was higher than that of some developing countries (5-10%) (Katouli et al., 1990); however, there are reports from Lao People's Democratic Republic (16.8%) and Tanzania (12.2%), indicating similar high frequencies (Gascon et al., 2000). The rate of isolation of Shigella spp., in developed countries is about 1% (Essers et al., 2000).
According to the results, the distribution of Shigella spp., in male more than in female represent 54.54 and 45.46% respectively. In this study we found that the incidence of diarrhea in male is almost similar to female. However recent studies showed that no sex difference in the occurrences of diarrhea was found as the risk factors associated with diarrhea are environmental and sociodemographic rather than biological factors (Parashar et al., 1998).     The first age group (1 month-10 year showed the high number of isolates (9 isolates), while the lowest incidence were in (10-20 year) and (60-70 year) one isolate to both of them as shown in Table 2. The average age of the patients with Shigella infection in our study was similar to some reports (MoezArdalan et al., 2003), indicating a rise in the average age of Shigella infection to older (aged 12 or more than 12 years) compared to some reports from other developing countries, where the group of one or more than one to less than five year (s) of age had the highest frequency of isolation (Ahmed et al., 1997). While (Rawashdeh et al., 1994) noticed that the age group for peak Shigella incidence was 1-4 years.
Shigella flexneri was the most predominant Serogroups (72.7%) followed Shigella boydii (18.2%) and Shigella dysentriae being the least common isolate (9.1%) ( Table 3). These results were agreed with the finding (Chuang et al., 2006) they showed that S. flexneri was the most prevalent serotype (73.3%) followed by S. sonnei (26.5%).while the results of some of studies that have been done in Iran, Israel, the United States, Canadaand other developed countries. Revealed that S. sonnei is the predominant species in those countries and is more common in children than in adults. However, in Taiwan and Bangladesh the infections are mostly caused by S. flexneri. It has been suggested that factors involved in natural selection may have been the main reason for these discrepancies (Farshad et al., 2006). Though it is in contrast to the finding of developed world but is similar to that in other countries where diarrheal diseases are endemic too (Wasfy et al., 2000).

DISCUSSION
The results of the antibiotic susceptibility tests for four species of Shigella isolates are shown in Table 3. In total, of the 11 isolates, 18.18% (2 isolates) were resistant to Ciprofloxacin, 36.36% (4 isolates) were resistant to Chloramphenicol, 54.54% (6 isolates)were resistant to Cefotaxime, Cefepime, Cefixime and Ceftazidium, 63.63% (7 isolates) were resistant to Azithromycinand 72.72% (8 isolates) were resistant to Tetracycline and Erythromycin. Resistance to Pipracillin, Amoxacillin, Cephalothin, Kanamycin and Rifampicin was 100% detected. These results agree with (MoezArdalan et al., 2003) The sowed the most common resistance among Shigella spp., was to tetracycline (73.5%), trimethoprim-sulphamethoxazole (70.4%) and amoxicillin-clavulanic acid (50.0%). S. flexneri isolates were most frequently resistant to tetracycline (82.2%), amoxicillin-clavulanic acid (82.2%). And disagree with (Banajeh et al., 2001) the noticed most of the Shigella isolates were susceptible to nalidixic acid and cefotaximeand resistant to the other antibiotics. Mandomando et al. (2009) showed that Shigella isolates are resistant mostly to the most available, inexpensive antibiotics by various molecular mechanisms but remain susceptible to ciprofloxacin, which is the first line for empirical treatment of shigellosis in the country.
The results of the antimicrobial susceptibility tests showing a relatively higher number of multidrugresistant isolates and especially the emergence of resistance to Aminoglycosides and third-generation cephalosporins indicates that designing a surveillance system for antimicrobial resistance in Iraq and the introduction of integrated guidelines for the appropriate use of antibiotics are urgently needed. Multiple resistances with the patterns of Pipracillin, Amoxacillin, Cephalothin, Kanamycin and Rifampicin noticed in all the isolates are shown in Table 4. S. flexneri were showed multiple resistance ranging from 7-13 antibiotic, most of these isolates resistant 12-13 antibiotics.
In the other hand Shigella boydii and Shigella dysentriae showed low resistance pattern they were 6 and 7, respectively. According to the susceptibility of the majority of Shigella spp., to Ciprofloxacin and Chloramphenicole in this study, recommend the morereadily available drug. Chu et al. (1998) indicated multiresistance (resistance to four or more agents) was more common in S. flexneri than in Shigella sonnei. A higher multidrug-resistant rate among Shigella isolates was found in our study. Although only limited numbers of strains were examined, this result may showed a clear trend that multidrug-resistant Shigella in Asian countries has been increasingand this may be due to a worsening situation with regard to antibiotic overuse in both humans and animals (Kuo et al., 2008). To reveal whether the multiple drug resistance phenomenons in the Shigella spp., was plasmid mediated, 11 Shigella spp., isolates were screened for plasmid. Numerous plasmid patterns were found in the isolates. Representative plasmid profiles from each Shigella species are shown. Many isolates demonstrated the large virulence plasmid (180-220 kb). This plasmid was not used for pattern comparison of isolates because of its documented instability on subculture (Sansonetti et al., 1981;Shafik et al., 2007). All the Shigella spp., isolates were found to harbour a single and similar plasmid except isolates 4 and 8 had which in two plasmids bands are included in Fig. 1 and Table 1 for comparison purposes. Plasmid fingerprinting is a helpful tool in epidemiological studies, particularly if there is a spectrum of plasmid profiles in the population. In our study, numerous plasmid patterns were found in each of the Shigella species. Tacket et al. (1984) also found multiple plasmid profiles in all Shigella species. Litwin et al. (1991) studied 74 Shigella spp., isolates, they found Plasmid patterns for each species were distinct. A total of 57 of 74 (77%) Shigella flexneri strains could be placed into seven plasmid patterns, 70 of 79 (89%) Shigella sonnei strains could be placed into seven patterns, 12 Shigella boydii strains could be placed into six patternsand each of 3 Shigella dysenteriae strains differed.
It was found this study there were relation between plasmid bands and multiple antibiotic resistant pattern. As shown in (Table 4). These results agree with Haider et al. (1985) who found that multiresistant clinical isolates generally harbor a single large transmissible plasmid.
Studies demonstrating the relation of plasmid and drug resistance in clinical isolates of Shigella spp., by curing are scanty in our country. Curing of plasmids was carried out with ethidium bromide, sub culture and temperature at 15-30°C. Plasmid curing was achieved by growing the strains, treatment with temperature at 15-30°C and Ethidium bromide, while the lowest curing efficiencies was obtained using subculture as shown in Table 5-7. The plasmid elimination was accompanied by drastic changes in antibiotic resistance and morphology of the colonies (Raja and Selvam, 2009). All isolates those were resistant to ciprofloxacin and chloramphenicol, lose resistance when treated with temperature at 15 and increase inhibition zone diameter when treated with temperature at 30°C.while in sub culturing and Ethidium bromide treatment chloramphenicol resistance remained, as well as isolates gave low resistant (inhibition zone) to ciprofloxacin. In another hand third generation cephalosporin affected by both curing treatment temperature at 15-30°C and Ethidium bromide. The results showed isolates death in ethidium bromide at 6.25 concentrations.
After curing experiments the loss of antibiotic resistance was concomitant with the loss of plasmid content so that the results showed that most cured isolates had lost their antibiotic resistance to almost antibiotics tested. This indicates that the resistance determinants of tested antibiotics were located on plasmids. Furthermore, this study suggested that loss of antibiotic resistance phenotype in cured strains may be either because of mutation as a result of incubation in the presence of the curing, or genes encoding resistance to the antibiotics.

CONCLUSION
Approximately 89.5% of the diarrhea cases had no bacterial pathogen, suggested of probability of viral infection. Similar to other studies (Patel et al., 2008) which indicated that 84% of diarrheal patients caused by other microorganism than other bacteria.