zahra safiri

Shigella species are among the common causes of bacterial diarrhoeal diseases. Traditional detection methods are time-consuming resulting in delay in treatment and control of Shigella infections thus there is a need to develop molecular methods for rapid and simultaneous detection of Shigella spp. In this study a rapid multiplex PCR were developed for simultaneous detection of three pathogenic Shigella species. For detection of Shigella spp., a pair of primers was used to replicate a chromosomal sequence. Three other sets of primers were also designed to amplify the target genes of three most common species of Shigella in Iran including S. sonnei, S. flexneri and S. boydii. The multiplex PCR assay was optimized for simultaneous detection and differentiation of three pathogenic Shigella species. The assay specificity was investigated by testing different strains of Shigella and other additional strains belonging to non Shigella species, but responsible for foodborne diseases. The Shigella genus specific PCR yielded the expected DNA band of 159 bp in all tested strains belonging to four Shigella species. The standard and multiplex PCR assays also produced the expected fragments of 248 bp, 503 bp, and 314 bp, for S. boydii, S. sonnei and S. flexneri, respectively. Each species-specific primer pair did not show any cross-reactivity. Both standard and multiplex PCR protocols had a good specificity. They can provide a valuable tool for the rapid and simultaneous detection and differentiation of three most prevalent Shigella species in Iran.

Direct electron transferring of glucose oxidase was investigated on reduced graphene and graphene oxide templates. The direct electrochemistry glucose oxidase on graphene showed a cyclic voltammograms corresponding to the FAD/FADH2 redox couple with an anodic, cathodic and formal potential of -430, -460 and -445 mV, respectively in 0.1 M phosphate buffer solution and air saturated condition for similarity of in vivo usage. The cyclic voltammograms of glucose oxidase on graphene is reversible. Also, the voltammograms results show, the current intensity of glucose oxidase on graphene is high, due to fast electron transferring. Moreover, the linear rang concentration of glucose on graphene are 0.4–9 µM. These studies make useful insight into the enzyme immobilization on nanoparticles for biosensors and bio-fuel cell preparation.

Introduction and Salmonellosis is responsible for large numbers of infections in both humans and animals. Conventional methods of isolation of Salmonella strains take 4-7 days to complete and are therefore laborious and require substantial manpower. Our main objective was to develop a rapid detection method using shortened PCR cycles in a conventional thermal cyclers and fast electrophoresis for Salmonella. The PCR primers for tyv (rfbE), prt (rfbS) and invA, genes were used for the rapid identification of S. enterica serovars typhi and paratyphi A, with rapid and short cycles of multiplex PCR. By using very fast and simple DNA extraction method in 10mins, rapid PCR cycles with total times of 35mins and rapid electrophoresis procedure with simple and very cheap buffer in 15mins we were able to separate the PCR products. All references and clinical isolates of Salmonella serovars typhi and paratyphi were accurately identified. Specificity analysis revealed no cross reaction with other enterobacterial strains. The sensitivity of the assay was 1-10 cells. The total time of multiplex PCR from sample preparation to final result is 45 to 50mins. These data indicate that the specificity and sensitivity of optimized rapid cycle multiplex PCR is a potentially valuable tool for rapid diagnosis of S. typhi using a conventional thermal cycler. This method cut the time of a PCR reaction from 3.5h to less than 60mins. These findings could also be applied to other PCR programs detecting various genes allowing researchers to significantly shorten their PCR reaction times.

Shigellosis is one of the major causes of morbidity in children with diarrhea in Iran. Integrons play an important role in the evolution and dissemination of multidrug resistance in gram-negative bacteria. The occurrence of integrons among Shigella spp. is frequently reported throughout the world. The aim of this study was to assess the occurrence of class 2 integrons among the multi drug resistant S. sonnei isolated from Iranian children in 2005. The study was conducted in two major pediatric hospitals in Tehran, Iran. Fecal specimens and rectal swab collected from patients were cultured and identified as Shigella by the conventional methods. Antimicrobial susceptibility test was performed according to the standard CLSI guideline. Multi-drug resistant isolates of S. sonnei were further examined for the presence of class 2 integron by PCR using specific primers. Amplicons were confirmed by restriction endonuclease analysis. A total of 83 multi-drug resistant S. sonnei strains were isolated. Of these, 45 (54%) exhibited a class 2 integron of 2.1 kbp, and 34 (41%) a class 2 integron of 1.3 kbp. Class 2 integrons were not detected in four isolates. The results showed an increased occurrence of class 2 integron carrying S. sonnei isolated from children in Tehran in 2005.

Fluoroquinolones, in particular ciprofloxacin have recently become the drug of choice for antibiotic therapy of invasive Salmonella infections in many countries. however, several treatment failures due to Salmonella strains with decreased susceptibility to ciprofloxacin have been reported. These isolates are still considered to be susceptible to ciprofloxacin according to CLSI interpretive criteria (MIC≤1 μg/ml) while they can not be detected by disk diffusion test. CLSI has recommended nalidixic acid disk diffusion as an indicator test for screening of Salmonella isolates with decreased susceptibility to ciprofloxacin.In this study the value of nalidixic acid susceptibility testing for screening of clinical strains of Salmonella with decreased susceptibility to ciprofloxacin was evaluated. Salmonella spp. strains have been isolated from several provinces in Iran during 2007-2008. The strains were identified by standard biochemical tests and serology. The susceptibility of the isolates to nalidixic acid and ciprofloxacin was determined by disk diffusion method. MIC assay for ciprofloxacin was determined by E. test. 28 out of 53 Salmonella strains (52.8%) were resistant to nalidixic acid. Disk diffusion testing showed that all isolates were susceptible to ciprofloxacin however 8 (15%) resistant nalidixic acid isolates showed decreased susceptibility to ciprofloxacin (MIC= 0.125 μg/ml) when tested by E.test. The results showed that nalidixic acid disk diffusion test can be considered as a reliable and costeffective method for screening of Salmonella strains with low level resistance to ciprofloxacin.