Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via S. Chiara 27, 61029 Urbino, Italy.
Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy.
Int J Mol Sci. 2023 Jul 18;24(14):11618. doi: 10.3390/ijms241411618.
Persistent and viable but non-culturable (VBNC) cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated -targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.
持续存在但不可培养（VBNC）细胞主要负责囊性纤维化（CF）肺部感染的复发和对抗生素的不反应性。在连续治疗周期之间在 CF 肺部中发现的亚抑制抗生素浓度可引发进入 VBNC 状态，尽管具有菌株特异性模式。在这里，我们分析了在暴露于饥饿与/或抗生素的情况下，两种 CF 分离株的生物膜中 VBNC 细胞的诱导，并研究了所涉及的潜在遗传决定因素。通过经过验证的靶向 qPCR 方案定量了总活菌细胞，并将 VBNC 细胞估计为 qPCR 和培养计数之间的差异。对分离株进行了全基因组测序，重点关注其携带的氨基糖苷类抗生素耐药基因以及鉴定突变的毒素-抗毒素和系统。获得的结果表明，不同抗生素耐药机制对 VBNC 细胞丰度的贡献不同，确定了妥布霉素外排的主要贡献，这是由 MexXY 外排泵过度表达介导的。基因组分析表明存在潜在的突变热点，值得进一步研究。因此，药物外排可能是进入 VBNC 状态的关键机制，也是抗持久性策略的潜在目标。
