多重抗藥性菌株Citrobacter werkmanii JA3，是從台灣中部醫院住院病人所分離的臨床菌株。根據等電點聚焦電泳實驗，顯示C. werkmanii JA3帶有pI 7.4及pI值為約9的乙內醯胺酶。使用質體DNA進行聚合酶連鎖反應及定序，發現C. werkmanii JA3質體上帶pI值約為9的ampC基因；而在細菌接合試驗中，也證實了此乙內醯胺酶的基因，位在於質體上，接合轉形株J53（pCW）也呈現多重抗藥性。質體pCW帶有2264-bp的Class I integron，其基因卡匣帶有drfA12和aadA2基因。選殖ampC 基因得到帶有4075 bp片段的pBK-CW3質體，定序此片段，發現ampC上游帶有ampR，下游為4-diphosphocytidyl-2-C-methyl-D-erythritol（CDP-ME）kinase（ispE）、putative transcriptional regulator（pobR）和outer membrane lipoprotein（blc）。在DNA序列比對中，C. werkmanii JA3之ampC和
C. werkmanii染色體上的ampC最為相近，有95%的相似度；其上游的ampR基因，與E. coli KU6400的ampR基因在DNA序列上也有92%的相似度。綜合以上的結果，都可推測此質體上的乙內醯胺酶基因是由染色體易位而來。在乙內醯胺酶活性測試中，ampC的乙內醯胺酶表現是持續性且依賴AmpR蛋白。聚合酶連鎖和定序反應，發現C. werkmanii JA3染色體上帶有ampD基因，DNA序列比對後與C. freundii OS60之ampD最為相近有90%的相似度。在親源關係比對，顯示 C. werkmanii JA3之AmpC、AmpR及AmpD，分別與C. werkmanii之AmpC；E. coli KU6400之AmpR；C. freundii OS60之AmpD最為相近。最後，本研究發現了檸檬酸桿菌屬的臨床菌株
C. werkmanii JA3位於質體的新型的AmpC 乙內醯胺酶其上游帶有
AmpR。
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.
