Beyond the emergence of plasmid-encoded mechanisms, mutation within the pmrAB genes remains one of the primary colistin resistance mechanisms in Escherichia coli. However, the mechanisms of high-level colistin resistance (HLCR) have not been elucidated. In this study, we evaluated the HLCR mechanisms in five colistin-susceptible Avian pathogenic Escherichia coli (APEC) isolates after colistin exposure. Three PmrB substitutions (G19R, L167P, V88E) and two PmrB sequence duplication (PmrB-sd) mutations (68-77dup and 94-156dup) were detected. Chromosomal replacement and deletion mutagenesis revealed the two PmrB-sd mutations contribute to, but are not fully responsible for, HLCR in APEC strains. Quantitative reverse transcription/polymerase chain reaction (qRT-PCR) revealed that the PmrB-sd induction mutants showed an increased pmrAB transcript level and the PmrB-sd reversion mutants exhibited a reduction of pmrAB expression. All five induction mutants exhibited decreased minimum inhibitory concentrations to florfenicol and tetracycline. In addition, four mutants (G19R, L167P, V88E, and 94-156dup) and two mutants (68-77dup and 94-156dup) also displayed increased sensitivity to ceftiofur and gentamicin, respectively. Zeta potential measurement of the induction mutants showed that there was less negative charge on the cell surface compared with its parental strains in the absence of colistin. The induction mutants also showed an increase of lag time and decrease of fitness. In summary, the identification of novel PmrB-sd mutations contributing to HLCR is helpful to broaden the knowledge of colistin resistance. Attention should be paid to the use of colistin for the treatment of infections caused by APEC strains.

中文翻译：

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pmrB 基因内的序列重复有助于禽致病性大肠杆菌的高水平粘菌素抗性。

除了质粒编码机制的出现之外，pmrAB基因内的突变仍然是大肠杆菌中主要的粘菌素耐药机制之一。然而，高水平粘菌素耐药性（HLCR）的机制尚未阐明。在这项研究中，我们评估了五种对粘菌素敏感的禽致病性大肠杆菌的 HLCR 机制(APEC) 在粘菌素暴露后分离。检测到三个 PmrB 替换（G19R、L167P、V88E）和两个 PmrB 序列重复（PmrB-sd）突变（68-77dup 和 94-156dup）。染色体置换和缺失诱变揭示了两个 PmrB-sd 突变有助于但不完全负责 APEC 菌株中的 HLCR。定量逆转录/聚合酶链反应 (qRT-PCR) 显示 PmrB-sd 诱导突变体显示pmrAB转录水平增加，而 PmrB-sd 回复突变体显示pmrAB减少表达。所有五个诱导突变体都表现出对氟苯尼考和四环素的最小抑制浓度降低。此外，四个突变体（G19R、L167P、V88E 和 94-156dup）和两个突变体（68-77dup 和 94-156dup）也分别显示出对头孢噻呋和庆大霉素的敏感性增加。诱导突变体的 Zeta 电位测量表明，与不存在粘菌素的亲本菌株相比，细胞表面的负电荷更少。诱导突变体还表现出滞后时间的增加和适应性的降低。总之，鉴定导致 HLCR 的新型 PmrB-sd 突变有助于拓宽对粘菌素耐药性的认识。应注意使用粘菌素治疗APEC菌株引起的感染。