Molecular characterization of multiple drug resistance in clinical isolate of Citrobacter werkmanii.

• Main Authors: Huei-Fen Chen, 陳惠芬
• Other Authors: 吳禮字
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/61501501155465799995

碩士 === 中國醫藥大學 === 基礎醫學研究所 === 98 === A multiple drug resistant strain of Citrobacter werkmanii JA3, was isolated from an adult patient hospitalised in Taichung, Taiwan. Isoelectric focusing revealed two beta -lactamases with isoelectric points of 7.4 and 9. Following PCR with plasmid DNA templates and gene sequencing, an enzyme was shown to correspond to AmpC-like enzyme with pI 9. The bla gene for ampC was transferable in conjugation experiments, resulting in a similar resistance profile to that observed in the original donor strain. Nucleotide sequence analyses a 2264-bp integron fragment from plasmid which encoded the class 1 integron with gene cassettes drfA12 and aadA2. The resistance gene of ampC which was cloned and expressed in E. coli, proved to contain an open reading frame showing 95% DNA sequence identity with the ampC gene in chromosome of C. werkmanii, DNA sequence analysis also identified a gene upstream of ampC whose sequence was 92% identical to the ampR gene from E. coli KU6400. In addition, a 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (ispE), putative transcriptional regulator (pobR) and an outer membrane lipoprotein (blc) in the dowmstream of ampC genes in plasmid pBK-CW3 with 4075 bp insert were identified. These results confirm the evidence of the translocation of a beta-lactamase-associated gene region from the chromosome to a plasmid. Beta-lactamase assays suggested that expression of ampC gene was constitutive and dependent on AmpR. Furthermore, ampD gene of C. werkmanii JA3 was 90% identical to the ampD gene from C. freundii OS60. Phylogenetic analysis amino acid residues of AmpC, AmpR and AmpD suggested that are most closely related to the AmpC of C. werkmanii, AmpR of E. coli KU6400 and AmpD of C. freundii. Finally, we describe a novel plasmid-encoded AmpC beta-lactamase with an ampR gene derived from Citrobacter spp. in Taiwan.