Servicio de Microbiología and Unidad de Investigación, Hospital Universitario Son Espases, Palma de Mallorca, Spain.
Antimicrob Agents Chemother. 2012 Sep;56(9):4771-8. doi: 10.1128/AAC.00680-12. Epub 2012 Jun 25.
We investigated the mechanisms leading to Pseudomonas aeruginosa pan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence of oprD, ampD, and dacB mutations was explored by PCR followed by sequencing and the expression of ampC and efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC(50)s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressed ampC and one or several efflux pumps, particularly mexB and/or mexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n = 1) or diminished expression (n = 1) of PBP1a and a lack of PBP4 expression (n = 4), which correlated with AmpC overexpression driven by dacB mutation. Analysis of the essential PBP IC(50)s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC(50)s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in the in vivo emergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.
我们研究了铜绿假单胞菌泛β-内酰胺耐药（PBLR）在治疗医院获得性感染过程中产生的机制，特别关注青霉素结合蛋白（PBP）谱的变化以及亚胺培南、头孢他啶和头孢洛扎烷（前 CXA-101）与 PBP 结合亲和力的变化。为此，我们研究了 6 对克隆相关的连续敏感-PBLR 分离株。通过 PCR followed by sequencing 探索 oprD、ampD 和 dacB 突变的存在，并通过实时逆转录-PCR 检测 ampC 和外排泵基因的表达。使用荧光青霉素 Bocillin FL 测定所有配对物膜制剂中的 PBP 谱，并在其中 3 对中分析头孢洛扎烷、头孢他啶和亚胺培南的 50%抑制浓度（IC50）。尽管（0 到 5 倍稀释）观察到一定程度的增加，但所有 PBLR 分离株的头孢洛扎烷 MIC 均≤4μg/ml。所有 6 个 PBLR 分离株均缺乏 OprD，并过度表达 ampC 和一个或多个外排泵，特别是 mexB 和/或 mexY。此外，其中 5 个分离株显示出 PBP 谱的改变，包括 PBP1a 的改变模式（n=1）或表达减少（n=1）和 PBP4 表达缺失（n=4），这与 dacB 突变驱动的 AmpC 过度表达相关。对必需 PBP IC50 的分析显示，PBP1a/b 结合亲和力在每个敏感-PBLR 对和不同对之间均存在显著差异。此外，尽管基因表达或序列没有显著差异，但在 PBLR 分离株中观察到 PBP2（亚胺培南）和 PBP3（头孢他啶、头孢洛扎烷、亚胺培南）IC50 增加的明显趋势。因此，我们的结果表明，除了 AmpC、外排泵和 OprD 之外，PBP 模式的改变似乎在体内产生 PBLR 菌株的过程中起作用，这些菌株仍然保留对新型抗假单胞菌头孢菌素头孢洛扎烷的一定敏感性。
