Attalla Eriny T, Khalil Amal M, Zakaria Azza S, Evans Rhiannon, Tolba Nesrin S, Mohamed Nelly M
Microbiology and Immunology Department, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.
Quadram Institute Bioscience, Norwich, UK.
Gut Pathog. 2024 Dec 3;16(1):73. doi: 10.1186/s13099-024-00667-z.
Colistin resistance significantly constrains available treatment options and results in the emergence of pandrug-resistant (PDR) strains. Treating PDR infections is a major public health issue. A promising solution lies in using colistin-based combinations. Despite the availability of in vitro data evaluating these combinations, the in vivo studies remain limited.
Thirty colistin-resistant Klebsiella pneumoniae (ColRKp) isolates were collected from hospitalized patients. Colistin resistance was detected using broth microdilution, and antimicrobial susceptibility was tested using the Kirby-Bauer method against 18 antibiotics. Extremely high resistance levels were detected, with 17% of the isolates being PDR. Virulence profiling, assessed using Anthony capsule staining, the string test, and the crystal violet assay, indicated the predominance of non-biofilm formers and non-hypermucoid strains. The isolates were screened for mcr genes using polymerase chain reaction. Whole-genome sequencing (WGS) and bioinformatics analysis were performed to characterize the genomes of PDR isolates. No plasmid-borne mcr genes were detected, and WGS analysis revealed that PDR isolates belonged to the high-risk clones: ST14 (n = 1), ST147 (n = 2), and ST383 (n = 2). They carried genes encoding extended-spectrum β-lactamases and carbapenemases, bla and bla, on conjugative IncHI1B/IncFIB plasmids, illustrating the convergence of virulence and resistance genes. The most common mechanism of colistin resistance involved alterations in mgrB. Furthermore, deleterious amino acid substitutions were also detected within PhoQ, PmrC, CrrB, ArnB, and ArnT. Seven colistin-containing combinations were compared using the checkerboard experiment. Synergy was observed when combining colistin with tigecycline, doxycycline, levofloxacin, ciprofloxacin, sulfamethoxazole/trimethoprim, imipenem, or meropenem. The efficacy of colistin combined with either doxycycline or levofloxacin was assessed in vitro using a resistance modulation assay, and in vivo, using a murine infection model. In vitro, doxycycline and levofloxacin reversed colistin resistance in 80% and 73.3% of the population, respectively. In vivo, the colistin + doxycycline combination demonstrated superiority over colistin + levofloxacin, rescuing 80% of infected animals, and reducing bacterial bioburden in the liver and kidneys while preserving nearly intact lung histology.
This study represents the first comparative in vitro and in vivo investigation of the efficacy of colistin + doxycycline and colistin + levofloxacin combinations in clinical PDR ColRKp isolates characterized at a genomic level.
对黏菌素的耐药性严重限制了可用的治疗选择，并导致泛耐药（PDR）菌株的出现。治疗PDR感染是一个重大的公共卫生问题。一个有前景的解决方案是使用基于黏菌素的联合用药。尽管有评估这些联合用药的体外数据，但体内研究仍然有限。
从住院患者中收集了30株对黏菌素耐药的肺炎克雷伯菌（ColRKp）分离株。使用肉汤微量稀释法检测黏菌素耐药性，并采用 Kirby-Bauer 法针对18种抗生素测试抗菌敏感性。检测到极高的耐药水平，17%的分离株为PDR。使用安东尼荚膜染色、拉丝试验和结晶紫试验评估毒力特征，结果表明非生物膜形成菌和非高黏液型菌株占主导。使用聚合酶链反应对分离株进行mcr基因筛查。进行全基因组测序（WGS）和生物信息学分析以表征PDR分离株的基因组。未检测到质粒携带的mcr基因，WGS分析表明PDR分离株属于高风险克隆：ST14（n = 1）、ST147（n = 2）和ST383（n = 2）。它们在接合性IncHI1B/IncFIB质粒上携带编码超广谱β-内酰胺酶和碳青霉烯酶bla和bla的基因，这说明了毒力和耐药基因的汇聚。黏菌素耐药的最常见机制涉及mgrB的改变。此外，在PhoQ、PmrC、CrrB、ArnB和ArnT中也检测到有害的氨基酸取代。使用棋盘试验比较了七种含黏菌素的联合用药。当黏菌素与替加环素、强力霉素、左氧氟沙星、环丙沙星、磺胺甲恶唑/甲氧苄啶、亚胺培南或美罗培南联合使用时观察到协同作用。使用耐药调节试验在体外评估黏菌素与强力霉素或左氧氟沙星联合使用的疗效，并在体内使用小鼠感染模型进行评估。在体外，强力霉素和左氧氟沙星分别使80%和73.3%的群体中的黏菌素耐药性逆转。在体内，黏菌素+强力霉素组合显示出优于黏菌素+左氧氟沙星组合，挽救了80%的感染动物，并降低了肝脏和肾脏中的细菌生物负荷，同时使肺组织学几乎保持完整。
本研究首次对黏菌素+强力霉素和黏菌素+左氧氟沙星组合在基因组水平表征的临床PDR ColRKp分离株中的疗效进行了体外和体内比较研究。
