Mangiaterra Gianmarco, Cedraro Nicholas, Vaiasicca Salvatore, Citterio Barbara, Galeazzi Roberta, Laudadio Emiliano, Mobbili Giovanna, Minnelli Cristina, Bizzaro Davide, Biavasco Francesca
Department of Life and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche, 60131 Ancona, Italy.
Department of Biomolecular Science, Biotechnology Section, University of Urbino "Carlo Bo", via Arco d'Augusto 2, 61032 Fano, Italy.
Antibiotics (Basel). 2020 Jul 10;9(7):399. doi: 10.3390/antibiotics9070399.
The recurrence of (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.
（绿脓杆菌）生物膜感染的复发是囊性纤维化（CF）患者的一个主要问题。抗生素无反应性持留菌和/或活的但不可培养（VBNC）形式的存在起着关键作用，亚抑菌浓度的抗生素可能有利于它们的形成。在绿脓杆菌生物膜模型中，研究了广泛用于治疗CF绿脓杆菌肺部感染的妥布霉素和环丙沙星对VBNC细胞数量的影响。在实验室菌株PAO1-N和临床菌株C24的体外生物膜中，将其在未添加或添加四分之一最小抑菌浓度（MIC）的妥布霉素或环丙沙星的无营养（NN）肉汤中传代培养170天以使其饥饿。通过qPCR和流式细胞术检测的总活菌数与菌落形成单位（CFU）计数之间的差异来估计VBNC细胞数量，结果显示出菌株和药物特异性模式。在所有条件下均检测到PAO1-N菌株的VBNC细胞数量更多且出现得更早。将C24菌株暴露于NN和NN +环丙沙星仅诱导了一个短暂的VBNC亚群，而在暴露于妥布霉素的生物膜中，该亚群在实验结束前数量更多且更稳定。在PAO1-N菌株中也观察到了对妥布霉素的相同反应。这些发现表明，低浓度的妥布霉素可能通过促进VBNC形式的形成而导致绿脓杆菌感染复发。
