Guérin François, Isnard Christophe, Cattoir Vincent, Giard Jean Christophe
CHU de Caen, Service de Microbiologie, Caen, France Université de Caen Normandie, EA4655 (équipe "Antibio-résistance"), Caen, France.
CHU de Caen, Service de Microbiologie, Caen, France Université de Caen Normandie, EA4655 (équipe "Antibio-résistance"), Caen, France
Antimicrob Agents Chemother. 2015 Dec;59(12):7753-61. doi: 10.1128/AAC.01729-15. Epub 2015 Oct 5.
Enterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-β-D-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.
阴沟肠杆菌复合体（ECC）是一种机会致病菌，在全球范围内导致住院患者发生多种感染，它主要通过产生AmpCβ-内酰胺酶来抵抗β-内酰胺类抗生素。在某些β-内酰胺类抗生素存在的情况下，AmpC的表达是高度可诱导的，但其与肽聚糖循环密切相关的潜在基因调控机制仍知之甚少。在本研究中，我们构建了不同的突变菌株，这些菌株在编码怀疑参与该途径的酶的基因中受到影响。正如预期的那样，ampC、ampR（编码ampC的调节蛋白）和ampG（编码一种通透酶）的失活消除了β-内酰胺类抗生素抗性。逆转录定量PCR（qRT-PCR）实验与表型研究相结合表明，头孢噻肟（高浓度）和头孢西丁以不同方式诱导ampC的表达：一种涉及NagZ（一种N-乙酰-β-D-氨基葡萄糖苷酶），另一种独立于NagZ。与铜绿假单胞菌建立的模型不同，DacB（也称为PBP4）的失活不是ECC中组成型ampC过表达的原因，而它导致了AmpC介导的高水平β-内酰胺类抗生素抗性，提示存在一种转录后调控机制。通过对dacB缺失突变体进行转录组测序（RNA-seq）的全局转录组分析证实了这些结果。最后，对37株ECC临床分离株的分析表明，与通常发现dacB突变的铜绿假单胞菌相反，AmpD序列中的氨基酸变化可能是体内高水平β-内酰胺类抗生素抗性发展中最关键的事件。这些发现为更好地理解ECC中的β-内酰胺类抗生素抗性带来了新的因素，这对于鉴定新潜在药物靶点至关重要。
