Health Protection Agency, Microbiology Services-Colindale, London, UK.
J Antimicrob Chemother. 2011 Sep;66(9):2006-10. doi: 10.1093/jac/dkr265. Epub 2011 Jul 8.
Two clinical isolates of Escherichia coli, EC18 and EC21, were non-susceptible (MICs 4-16 mg/L) to cefpirome and cefepime, with marked synergy with clavulanate, yet were susceptible to cefotaxime and ceftazidime (MICs ≤ 1 mg/L). EC19, from the same patient as EC21, was susceptible to all four cephalosporins. We sought to characterize the molecular basis of resistance in isolates EC18 and EC21.
PFGE was used to study the genetic relationships of the isolates, and MICs were determined. β-Lactamases were characterized by PCR, isoelectric focusing (IEF), construction of genomic libraries and sequencing. A double mutant of E. coli J53 was constructed, lacking OmpC and OmpF porins. Plasmids from clinical isolates were transformed into E. coli J53 and J53ΔompCF. Outer membrane proteins (OMPs) were analysed by SDS-PAGE and OmpA by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. Expression of omp and bla genes was analysed by RT-PCR.
Isolates EC19 and EC21 had identical PFGE profiles, whereas EC18 was distinct. PCR and IEF confirmed β-lactamases with pIs of 5.4 (TEM-1) in EC18 and 7.4 (OXA-1) in both EC19 and EC21. EC18 had bla(TEM-1b) with the strong promoter P5 and lacked OmpC and OmpF. RT-PCR showed stronger expression of bla(OXA-1) in EC21 versus EC19, along with diminished expression of OmpC, though with increased OmpF. Plasmids extracted from EC18 and EC21 conferred increased MICs of cefpirome and cefepime, although susceptibility to cefotaxime and ceftazidime was retained.
The 'cefpiromase' or 'cefepimase' ESBL phenotype of the clinical isolates non-susceptible to cefpirome and cefepime resulted from high expression of TEM-1 or OXA-1 β-lactamases combined with loss of porins.
两株临床分离的大肠杆菌 EC18 和 EC21 对头孢吡肟和头孢噻肟耐药（MIC 值为 4-16mg/L），与克拉维酸有明显协同作用，但对头孢噻肟和头孢他啶敏感（MIC 值≤1mg/L）。来自与 EC21 相同患者的 EC19 对所有四种头孢菌素均敏感。我们试图描述 EC18 和 EC21 分离株耐药的分子基础。
PFGE 用于研究分离株的遗传关系，测定 MIC 值。通过 PCR、等电聚焦（IEF）、基因组文库构建和测序来鉴定β-内酰胺酶。构建大肠杆菌 J53 的双突变体，缺乏 OmpC 和 OmpF 孔蛋白。将临床分离株的质粒转化入大肠杆菌 J53 和 J53ΔompCF。通过 SDS-PAGE 分析外膜蛋白（OMPs），基质辅助激光解吸电离飞行时间/飞行时间质谱法分析 OmpA。通过 RT-PCR 分析 omp 和 bla 基因的表达。
EC19 和 EC21 分离株的 PFGE 图谱相同，而 EC18 则不同。PCR 和 IEF 证实 EC18 中有 pI 为 5.4（TEM-1）的β-内酰胺酶，EC19 和 EC21 中有 pI 为 7.4（OXA-1）的β-内酰胺酶。EC18 含有 bla(TEM-1b)，其强启动子 P5 和缺乏 OmpC 和 OmpF。RT-PCR 显示 EC21 中 bla(OXA-1)的表达更强，而 OmpC 的表达减弱，尽管 OmpF 增加。从 EC18 和 EC21 中提取的质粒赋予了头孢吡肟和头孢噻肟更高的 MIC 值，尽管保留了对头孢噻肟和头孢他啶的敏感性。
对头孢吡肟和头孢噻肟不敏感的临床分离株表现出“头孢吡肟酶”或“头孢噻肟酶”ESBL 表型，这是由于 TEM-1 或 OXA-1β-内酰胺酶的高表达与孔蛋白的缺失相结合所致。
