Fernández-Billón María, Jordana-Lluch Elena, Llambías-Cabot Aina E, Gomis-Font María A, Fraile-Ribot Pablo, Torrandell Rosa I, Colman-Vega Pamela J, Murillo Óscar, Macià María D, Oliver Antonio
Department of Microbiology, Hospital Universitario Son Espases, Health Research Institute of the Balearic Islands (IdISBa), 07120 Palma de Mallorca, Spain.
Centro de Investigación Biomédica en Red en Enfermedades Infecciosas, Instituto de Salud Carlos III (CIBERINFEC), 28029 Madrid, Spain.
Biofilm. 2024 Oct 22;8:100231. doi: 10.1016/j.bioflm.2024.100231. eCollection 2024 Dec.
New combinations of β-lactams and β-lactamase inhibitors, such as ceftolozane/tazobactam could be useful to combat biofilm-driven chronic infections by extensively resistant (XDR) but resistance development by mutations in the Ω-loop of AmpC has been described. However, these mutations confer collateral susceptibility to carbapenems. Thus we aimed to evaluate the therapeutic efficacy and the prevention of resistance development of regimen alternating ceftolozane/tazobactam and imipenem.
A carbapenem-resistant XDR clinical strain (ST175, 104-B7) and its isogenic imipenem-susceptible ceftolozane/tazobactam-resistant mutant derivative (AmpC T96I, 104-I9) were used. Experiments of single strains and mixed (104-B7 and 104-I9, 1:0.01 ratio) biofilms were performed. 48h biofilms (flow cell system) were treated for 6 days with either ceftolozane/tazobactam, 4/4 mg/L or the alternation of ceftolozane/tazobactam (2 days)-imipenem 4 mg/L (2 days) - ceftolozane/tazobactam (2 days). After treatment, biofilms were collected and plated on Mueller-Hinton agar± ceftolozane/tazobactam 4/4 mg/L. Structural dynamics were monitored using confocal laser scanning microscopy and images were processed with IMARIS software. At least, three independent triplicate experiments per condition were performed. Emerging resistant mutants were characterized through whole genome sequencing (Illumina).
Ceftolozane/tazobactam monotherapy failed to reduce the biofilms of the 104-B7 XDR strain and led to the selection of resistant mutants that showed AmpC Ω-loop mutations (T96I, L244R or aa236Δ7). On the contrary, alternation with imipenem enhanced activity (3 Logs reduction at day 6) and prevented the emergence of ceftolozane/tazobactam-resistant mutants. Likewise, treatment with ceftolozane/tazobactam dramatically amplified the resistant strain 104-I9 in mixed biofilms (>90 % of the population), while the alternation regimen counterselected it.
Collateral susceptibility-guided alternation of ceftolozane/tazobactam with imipenem effectively prevented the selection of resistant mutants and thus could be a potential therapeutic strategy for the treatment of XDR chronic infections.
新型β-内酰胺类与β-内酰胺酶抑制剂组合，如头孢洛扎/他唑巴坦，可能有助于对抗由广泛耐药（XDR）菌形成生物膜驱动的慢性感染，但已有报道称AmpC的Ω环发生突变会导致耐药性产生。然而，这些突变使细菌对碳青霉烯类药物产生协同敏感性。因此，我们旨在评估头孢洛扎/他唑巴坦与亚胺培南交替用药方案的治疗效果及预防耐药性产生的作用。
使用一株耐碳青霉烯类的XDR临床菌株（ST175，104 - B7）及其等基因的对亚胺培南敏感但对头孢洛扎/他唑巴坦耐药的突变衍生物（AmpC T96I，104 - I9）。进行单菌株和混合（104 - B7和104 - I9，比例为1:0.01）生物膜实验。用头孢洛扎/他唑巴坦（4/4 mg/L）或头孢洛扎/他唑巴坦（2天）-亚胺培南4 mg/L（2天）-头孢洛扎/他唑巴坦（2天）交替方案处理48小时的生物膜（流动细胞系统）6天。处理后，收集生物膜并接种在含或不含头孢洛扎/他唑巴坦4/4 mg/L的穆勒 - 欣顿琼脂平板上。使用共聚焦激光扫描显微镜监测结构动态，并使用IMARIS软件处理图像。每种条件至少进行三个独立的重复实验。通过全基因组测序（Illumina）对新出现的耐药突变体进行表征。
头孢洛扎/他唑巴坦单药治疗未能减少104 - B7 XDR菌株的生物膜，并导致选择出显示AmpC Ω环突变（T96I、L244R或aa236Δ7）的耐药突变体。相反，与亚胺培南交替使用增强了活性（第6天减少3个对数）并防止了头孢洛扎/他唑巴坦耐药突变体的出现。同样，在混合生物膜中，用头孢洛扎/他唑巴坦处理显著扩增了耐药菌株104 - I9（占菌群的>90%），而交替用药方案则对其进行了反向选择。
基于协同敏感性指导的头孢洛扎/他唑巴坦与亚胺培南交替用药有效防止了耐药突变体的选择，因此可能是治疗XDR慢性感染的一种潜在治疗策略。
