Characterization of CTX - M ESBLs in Enterobacter cloacae , Escherichia coli and Klebsiella 1 pneumoniae

2 3 Background: A high rate of resistance to 3 rd generation cephalosporins among Enterobacteriaceae 4 isolates from Egypt has been previously reported. This study aims to characterize the resistance 5 mechanism (s) to extended spectrum cephalosporins among resistant clinical isolates at a medical 6 institute in Cairo, Egypt. 7 Methods: Nonconsecutive Klebsiella pneumoniae (Kp), Enterobacter cloacae (ENT) and E. coli (EC) 8 isolates were obtained from the clinical laboratory at the medical institute. Antibiotic susceptibility 9 was tested by CLSI disk diffusion and ESBL confirmatory tests. MICs were determined using broth 10 microdilution. Isoelectric focusing (IEF) was used to determine the pI values, inhibitor profiles, and 11 cefotaxime (CTX) hydrolysis by the β-lactamases. PCR and sequencing were performed using blaCTX12 M and ISEcp1specific primers, with DNA obtained from the clinical isolates. Conjugation 13 experiments were done to determine the mobility of blaCTX-M. 14 Results: All five clinical isolates were resistant to CTX, and were positive for ESBL screening. IEF 15 revealed multiple β-lactamases produced by each isolate, including a β-lactamase with a pI of 8.0 in 16 Kp and ENT and a β-lactamase with a pI of 9.0 in EC. Both β-lactamases were inhibited by clavulanic 17 acid and hydrolyzed CTX. PCR and sequence analysis identified blaCTX-M-14 in Kp and ENT and a 18 blaCTX-M-15 in EC. Both blaCTX-M-14 and blaCTX-M-15 were preceded by ISEcp1 elements as revealed by 19 partial sequence analysis of the upstream region of the blaCTX-M genes. blaCTX-M-15 was transferable but 20 not blaCTX-M-14. 21 Conclusion: This is the first report of CTX-M-14 in Kp and ENT isolates from Egypt, the Middle East 22 and North Africa. 23 24