Abstract：
The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum β-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-β-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.
摘要：
这项研究的目的是评估对替莫西林的耐药比例，替加环素,环丙沙星,和称为t2c2的氯霉素表型，该表型是由ramAR基因座内的突变引起的，该突变是在法国大学医院的三个重症监护病房中分离的3年的超广谱β-内酰胺酶-肠杆菌（ESBL-E）中分离的。对所有443ESBL-E进行了两种平行方法，包括：（i）替莫西林的最低抑制浓度，替加环素,环丙沙星,和氯霉素被确定，（ii）从Illumina测序平台获得的基因组进行分析，以确定多位点序列类型，抗性体,以及包括ramAR操纵子在内的几种tetR相关基因的多样性。在包括的443种ESBL-E菌株中，大肠杆菌分离株（n=194），肺炎克雷伯菌（n=122），发现阴沟肠杆菌复合体（Ecc）（n=127）。31种ESBL-E菌株(7%)，16肺炎克雷伯菌（13.1%），15例Ecc(11.8%)除了它们的ESBL谱外，还呈现了t2c2表型，而没有大肠杆菌呈现这些抗性。通过添加Phe-Arg-β-萘甲酰胺，t2c2表型总是可逆的，表明阻力结瘤泵在这些观察中的作用。与t2c2表型相关的突变仅限于RamR，ramAR基因间区(IR)，AcrRRamR中的突变由其DNA结合域内或蛋白质-底物相互作用的关键位点内的C-或N-末端缺失和氨基酸取代组成。ramARIR显示参与RamRDNA结合结构域的核苷酸取代。序列的这种多样性表明RamR和ramARIR代表细菌抗微生物抗性的主要遗传事件。在重症监护病房（ICU）住院的患者中，由传染病引起的死亡率很高。这些结果的一部分可以用抗生素耐药性来解释，这延误了适当的治疗。可转移的抗生素抗性基因是解释ICU中多药耐药（MDR）细菌高率的众所周知的机制。这项研究描述了染色体突变的患病率，这导致MDR细菌中额外的抗生素耐药性。超过12％的肺炎克雷伯菌和阴沟肠杆菌复杂菌株在ramAR基因座内出现突变，与称为AcrAB-TolC的外排泵和孔蛋白：OmpF的失调有关。这些失调导致抗生素产量增加，特别是替加环素，环丙沙星,和氯霉素与β-内酰胺的输入减少有关，尤其是替莫西林.转录调节因子如ramAR基因座内的突变在抗生素抗性传播中起主要作用,需要进一步探索。
