Abstract—Fourty one strains of ESBL producing P.aeruginosa which were previously isolated from burn patients in Kerman University general hospital, Iran were subjected to PCR, RFLP and sequencing in order to determine the type of extended spectrum β- lactamases (ESBL), the restriction digestion pattern and possibility of mutation among detected genes. DNA extraction was carried out by phenol chloroform method. PCR for detection of bla genes was performed using specific primer for each gene. Restriction Fragment Length Polymorphism (RFLP) for ESBL genes was carried out using EcoRI, NheI, PVUII, EcoRV, DdeI, and PstI restriction enzymes. The PCR products were subjected to direct sequencing of both the strands for identification of the ESBL genes.The bla CTX-M, bla VEB-1, bla PER-1, bla GES-1 , bla OXA-1 , bla OXA-4 and bla OXA-10 genes were detected in the (n=1) 2.43%, (n=41)100%, (n=28) 68.3%, (n=10) 24.4%, (n=29) 70.7%, (n=7)17.1% and (n=38) 92.7% of the ESBL producing isolates respectively. The RFLP analysis showed that each ESBL gene has identical pattern of digestion among the isolated strains. Sequencing of the ESBL genes confirmed the genuinety of PCR products and revealed no mutation in the restriction sites of the above genes. From results of the present investigation it can be concluded that bla VEB-1 and bla CTX-M were the most and the least frequently isolated ESBL genes among the P.aeruginosa strains isolated from burn patients. The RFLP and sequencing analysis revealed that same clone of the bla genes were indeed existed among the antibiotic resistant strains. Keywords—ESBL genes, PCR, RFLP, Sequencing, P.aeruginosa I. INTRODUCTION seudomonas aeruginosa is ranking second among gram negative hospital acquiring pathogens and one of leading cause of burn infections reported to the National Nosocomial Infection Surveillance System [1, 2]. The idea of eradication of P. aeruginosa from burn patients through intense antimicrobial therapy may lead to significant selection of F. Shacheraghi & H. Noveiri are with Department of Microbiology, Pasture Institute of Iran, Tehran, Iran. MR. Shakibaie is with Department of Microbiology, Kerman University of Medical Sciences, Kerman, Iran. Corresponding author: MR, Shakibaie Department of Microbiology, Kerman University of Medical Sciences, End of 22-Bahman BLVD, 76167-14111, Kerman, Iran. Work Phone: +98-341- 3221660-64. Mobile Phone: +98-913-140-8226, Fax: +98-341- 3221676. e-mail: mr_shakibaei@kmu.ac.ir resistance strains in burn unit of the hospitals [3]. One of the important features of these strains is resistant to multiple clinically important antibiotics like third generation of cephalosporins, imipenem and aztronam [4]. Many P.aeruginosa strains produces different class of extended spectrum β-lactamases (ESBLs) that enable bacterium to stand against extended –spectrum cephalosporins, such as cefotaxime, ceftriaxone and ceftazidime and have been reported with increasing frequency [5, 6]. ESBL mediate resistance to cephalosporin antibiotics and were first discovered in Europe in the early 1980s. They have become a widespread problem, particularly in Klebsiella pneumoniae, and increasingly in non-typhoid Salmonella species. The OXA-type ESBLs have been found mainly in P. aeruginosa isolates from Turkey and France [7]. Traditionally, ESBL enzymes have been derivatives of TEM and SHV parent enzymes. The last year, however, has seen an explosion of developments in ESBLs of non-TEM, non-SHV lineage in Europe. The CTX-M type ESBLs have become particularly widespread [8]. Bert et al., [9] detected bla PSE and bla OXA gene variants using PCR. The genotypes were distinguished by restriction of PCR products with endonucleases recognizing sites involved in point mutations. Jiang et al., [10] studied a total of 75 clinical isolates of P. aeruginosa. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs and bla VEB-3 was the most prevalent ESBL gene reported by the authors. Antibiotic susceptibility tests and PCR amplification of genes encoding class A (bla PSE-1 , bla PER-1 , bla VEB-1 , bla TEM , bla SHV , bla CTX-M and bla GES-1 ) and class D ß-lactamases (bla OXA-groupI , bla OXA-groupII and bla OXA-groupIII ) in P.aeruginosa were carried out by Lee et al., (11). In 64 (25.4%) isolates, structural genes for PSE-1 (6.3%), OXA-10 (13.1%), OXA-4 (4.3%), OXA-30 (2.0%), OXA-2 (2.3%) and OXA-17 (0.4%) were found, their distribution varied between provinces. None harboured bla PER-1 , bla VEB-1 , bla TEM , bla SHV , bla CTX-M and bla GES-1 . Similarly, PCR and sequence analysis revealed the presence of the bla CTX-M-1 , bla SHV-1 and bla TEM-116 genes in the P. aeruginosa and bla CTX-M-1 and bla SHV-1 in the Stenotrophomonas maltophilia strains [12]. Mirsalehian et al., [13] studied the prevalence of ESBLs and antimicrobial susceptibilities of P. aeruginosa isolated from burn patients in Tehran, Iran. It was found that 50 (74.62%), 33 (49.25%) and Molecular Identification of ESBL Genes bla GES-1, bla VEB-1, bla CTX-M bla OXA-1, bla OXA-4, bla OXA-10 and bla PER-1 in Pseudomonas aeruginosa Strains Isolated from Burn Patients by PCR, RFLP and Sequencing Techniques Fereshteh Shacheraghi, Mohammad Reza Shakibaie, Hanieh Noveiri P World Academy of Science, Engineering and Technology International Journal of Bioengineering and Life Sciences Vol:4, No:1, 2010 114 International Scholarly and Scientific Research & Innovation 4(1) 2010 scholar.waset.org/1307-6892/219 International Science Index, Bioengineering and Life Sciences Vol:4, No:1, 2010 waset.org/Publication/219