EA 7380 Dynamyc, Université Paris-Est Créteil, Ecole nationale vétérinaire d'Alfort, USC Anses, Créteil, France.
Unité de Bactériologie-Hygiène, Département de prévention, diagnostic et traitement des infections, Hôpital Henri Mondor, AP-HP, Créteil, France.
Front Cell Infect Microbiol. 2020 Oct 6;10:574028. doi: 10.3389/fcimb.2020.574028. eCollection 2020.
The complexity of biofilms constitutes a therapeutic challenge and the antimicrobial susceptibility of fungal-bacterial biofilms remains poorly studied. The filamentous fungus (Af) and the Gram-negative bacillus (Sm) can form biofilms and can be co-isolated from the airways of cystic fibrosis (CF) patients. We previously developed an biofilm model which highlighted the antibiosis effect of Sm on Af, which was dependent on the bacterial fitness. The aim of the present study was to investigate the susceptibility of Af and Sm in mono- or polymicrobial biofilms to five antimicrobial agents alone and in two-drug combinations. Af and Sm clinical reference strains and two strains from CF sputa were tested through a planktonic and biofilm approaches. Af, Sm, or Af-Sm susceptibilities to amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), levofloxacin (LVX), and rifampicin (RFN) were evaluated by conventional planktonic techniques, crystal violet, XTT, qPCR, and viable plate count. Af planktonic cells and biofilms in formation were more susceptible to AMB, ITC, and VRC than Af mature biofilms. Af mature biofilms were susceptible to AMB, but not to ITC and VRC. Based on viable plate count, a lower concentration of LVX and RFN was required to reduce Sm cell numbers on biofilms in formation compared with mature biofilms. The antibiosis effect of Sm on Af growth was more pronounced for the association of CF strains that exhibited a higher fitness than the reference strains. In Af-Sm biofilms, the fungal susceptibility to AMB was increased compared with Af biofilms. In contrast, the bacterial susceptibility to LVX decreased in Af-Sm biofilms and was fungal biomass-dependent. The combination of AMB (64 μg/mL) with LVX or RFN (4 μg/mL) was efficient to impair Af and Sm growth in the polymicrobial biofilm. Sm increased the Af susceptibility to AMB, whereas Af protected Sm from LVX. Interactions between Af and Sm within biofilms modulate susceptibility to antimicrobial agents, opening the way to new antimicrobial strategies in CF patients.
生物膜的复杂性构成了治疗上的挑战，真菌-细菌生物膜的抗微生物敏感性仍然研究甚少。丝状真菌 （Af）和革兰氏阴性杆菌 （Sm）可以形成生物膜，并且可以从囊性纤维化 （CF）患者的气道中共同分离出来。我们之前开发了一种生物膜模型，该模型突出了 Sm 对 Af 的抗生作用，该作用依赖于细菌的适应性。本研究的目的是研究单一或混合微生物生物膜中 Af 和 Sm 对五种抗菌药物的敏感性，以及两种药物联合使用的敏感性。通过浮游和生物膜方法测试了 Af 和 Sm 的临床参考株以及来自 CF 痰液的两种菌株。通过常规浮游技术、结晶紫、XTT、qPCR 和活菌平板计数评估了两性霉素 B（AMB）、伊曲康唑（ITC）、伏立康唑（VRC）、左氧氟沙星（LVX）和利福平（RFN）对 Af、Sm 或 Af-Sm 的敏感性。与成熟生物膜相比，Af 浮游细胞和正在形成的生物膜对 AMB、ITC 和 VRC 更敏感。成熟的 Af 生物膜对 AMB 敏感，但对 ITC 和 VRC 不敏感。基于活菌平板计数，与成熟生物膜相比，形成中的生物膜中需要较低浓度的 LVX 和 RFN 来减少 Sm 细胞数量。Sm 对 Af 生长的抗生作用在 CF 菌株的联合作用下更为明显，这些菌株表现出比参考菌株更高的适应性。在 Af-Sm 生物膜中，与 Af 生物膜相比，真菌对 AMB 的敏感性增加。相比之下，在 Af-Sm 生物膜中，细菌对 LVX 的敏感性降低，并且与真菌生物量有关。AMB（64μg/mL）与 LVX 或 RFN（4μg/mL）的联合使用可有效地损害多微生物生物膜中 Af 和 Sm 的生长。Sm 增加了 Af 对 AMB 的敏感性，而 Af 则保护 Sm 免受 LVX 的侵害。生物膜内 Af 和 Sm 之间的相互作用调节对抗微生物药物的敏感性，为 CF 患者开辟了新的抗菌策略。
