Author/Authors :
Tahmasebi Birgani ، Maryam Department of Medical Genetics - School of Medicine - Ahvaz Jundishapur University of Medical Sciences , Bijanzadeh ، Mahdi Department of Medical Genetics - School of Medicine, Cellular and Molecular Research Center - Ahvaz Jundishapur University of Medical Sciences , Ansari ، Hossein Department of Biotechnology - Islamic Azad University, Ahvaz Branch
Abstract :
Background: Acinetobacter baumannii is one themostdangerous microbes that is resistant to a wide range of prescribed antibiotics, thus development of more effective approaches is urgent. Recombinant protein vaccines have been recently proposed as a safe method to cope with infectious agents. In this approach, antigenic gene of a bacterium can be cloned in an expression vector to produce recombinant protein. Objectives: In this study, the prevalence of A. baumannii was determined in hospitalized samples and the antimicrobial susceptibility patterns of isolates were characterized for a variety of antibiotics. The ompA gene fromresistant isolates was then amplified and cloned in an expression vector to produce the recombinant OmpA protein in Escherichia coli. Methods: The clinical samples were collected from sputum, wounds, septicemia, and urinary tract infections at the intensive care unit (ICU) and different wards of hospitals and A. baumannii were identified, according to standard diagnostic tests. The antibiotic susceptibility patterns of the isolates were determined for 17 antibiotics. The ompA gene of A. baumannii was amplified using the polymerase chain reaction (PCR). The ompA-amplicon was cloned and sub-cloned in pTZ57RT and pET32a (+) vectors, respectively. Double digestion, DNA sequencing, and PCR were performed to confirm that ompA has been truly cloned. Using RT-PCR and SDSPAGE, the expression of recombinant OmpA was assessed in IPTG-induced E. coli. Results: Most of the A. baumannii were resistant to antibiotics cefepime, ceftazidime, ceftriaxone, aztreonam, amikacin, and gentamycin, and the least resistance was found towards colistin, ampicillin-sulbactam, and trimethoprim antibiotics. The ompA gene was amplified as 1112 bp amplicon, which was successfully cloned and sub-cloned in the pTZ57RT-T/A and pET32a (+) vectors. The presence of ~ 38 kDa band in SDS-PAGE showed that the recombinant pET32a-ompA was highly expressed in the host cells. Conclusions: The obtained data showed the high resistance of A. baumannii-infected isolates to the antibiotics. Besides, successful cloning and expression of rOmpA in the cells suggests that the antigenic properties of ompA gene may be considered in future researches.
