Cabot Gabriel, Bruchmann Sebastian, Mulet Xavier, Zamorano Laura, Moyà Bartolomé, Juan Carlos, Haussler Susanne, Oliver Antonio
Servicio de Microbiología and Unidad de Investigación Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
Helmholtz Center for Infection Research, Braunschweig, Germany.
Antimicrob Agents Chemother. 2014 Jun;58(6):3091-9. doi: 10.1128/AAC.02462-13. Epub 2014 Mar 17.
We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ΔmutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 μg/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 μg/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.
我们比较了野生型（PAO1）和突变体（PAOMS，ΔmutS）铜绿假单胞菌对头孢他啶、美罗培南、环丙沙星和头孢洛扎-他唑巴坦耐药性产生的动态变化及机制。在一式三份的实验中，将菌株与每种抗生素0.5至64倍MIC浓度孵育24小时。将显示生长的最高抗生素浓度的试管中的菌液重新接种到含有浓度高达64倍MIC的新鲜培养基中，连续培养7天。对每个步骤、每种抗生素和每个菌株的两个分离菌落评估其药敏谱和耐药机制。通过RNA测序（RNA-seq）对头孢洛扎-他唑巴坦耐药突变体进行全基因组分析，以进一步表征。头孢他啶产生高水平耐药的速度最快，其次是美罗培南和环丙沙星。用这些抗生素筛选出的突变体均未显示对头孢洛扎-他唑巴坦的交叉耐药性。另一方面，头孢洛扎-他唑巴坦耐药性的产生要慢得多，仅在突变体菌株中观察到高水平耐药。对头孢洛扎-他唑巴坦中度耐药（MIC为4至8μg/ml）的PAO1衍生物仅显示2至4个突变，这些突变决定了与严重适应性代价相关的全局多效性效应。高水平耐药（MIC为32至128μg/ml）的PAOMS衍生物显示45至53个突变。在所有突变体中均检测到全局基因表达谱的主要变化，但只有PAOMS突变体显示ampC过表达，这是由dacB或ampR突变引起的。此外，所有PAOMS突变体的AmpC保守残基中含有1至4个突变（F147L、Q157R、G183D、E247K或V356I）。互补研究表明，与野生型ampC相比，这些突变大大增加了头孢洛扎-他唑巴坦和头孢他啶的MIC，但降低了哌拉西林-他唑巴坦和亚胺培南的MIC。因此，对头孢洛扎-他唑巴坦高水平耐药性的产生似乎仅在铜绿假单胞菌突变体背景下有效发生，其中多个突变导致AmpC过表达和结构修饰。
