Safety and Efficacy of a Phage, kpssk3, in an in vivo Model of Carbapenem-Resistant Hypermucoviscous Klebsiella pneumoniae Bacteremia

Abstract

Antimicrobial resistance (AMR) is one of the most significant threats to global public health. As antibiotic failure is increasing, phages are gradually becoming important agents in the post-antibiotic era. In this study, the therapeutic effects and safety of kpssk3, a previously isolated phage infecting carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP), were evaluated in a mouse model of systemic CR-HMKP infection. The therapeutic efficacy experiment showed that intraperitoneal injection with a single dose of phage kpssk3 (1 × 107 PFU/mouse) 3 h post infection protected 100% of BALB/c mice against bacteremia induced by intraperitoneal challenge with a 2 × LD100 dose of NY03, a CR-HMKP clinical isolate. In addition, mice were treated with antibiotics from three classes (polymyxin B, tigecycline, and ceftazidime/avibactam plus aztreonam), and the 7 days survival rates of the treated mice were 20, 20, and 90%, respectively. The safety test consisted of 2 parts: determining the cytotoxicity of kpssk3 and evaluating the short- and long-term impacts of phage therapy on the mouse gut microbiota. Phage kpssk3 was shown to not be cytotoxic to mammalian cells in vitro or in vivo. Fecal samples were collected from the phage-treated mice at 3 time points before (0 day) and after (3 and 10 days) phage therapy to study the change in the gut microbiome via high-throughput 16S rDNA sequence analysis, which revealed no notable alterations in the gut microbiota except for decreases in the Chao1 and ACE indexes.

Characteristics of a Bacteriophage, vB_Kox_ZX8, Isolated From Clinical Klebsiella oxytoca and Its Therapeutic Effect on Mice Bacteremia

“…After 5 × 10 9 PFU of phage vB_Kox_ZX8 was IP injected into healthy mice, the phage titer in the blood was detected within 10 min, maintained at a high level at 30–90 min, gradually decreased, and disappeared after 6 h. In a study of Podoviridae phage kpssk3, which has 81% sequence similarity to vB_Kox_ZX8, Klebsiella phage kpssk3 showed a similar short residence time in mouse blood. After intraperitoneal injection of 10 8 PFU phage kpssk3 for 15 min, the blood titer of phages reached 10 5 PFU/mL, gradually decreasing after maintaining the high titer state for 4 h, and disappeared completely after 8 h ( Shi et al, 2021 ). In other studies of the Podoviridae phage, Klebsiella phage Kp_Pokalde_002 and Pseudomonas phage PEV20 had a longer residence time in mouse blood.…”

Whole-genome sequencing, annotation, and biological characterization of a novel Siphoviridae phage against multi-drug resistant Propionibacterium acne

“…The next day, this mixture was centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was passed through 0.22-μm PES needle filters. The phage was purified and separated by picking single-plaque with the double-layer agar method ( Shi et al, 2021 ). And the resulting isolation was stored at 4°C.…”

“…Three 3-μL aliquots of the high-titer phage preparations (10 10 ~ 10 13 PFU /mL) were pipetted on carbon-coated copper grids as instructed (Shi et al, 2021). After allowing the phages 10.…”

Berberine Improves Vascular Dysfunction by Inhibiting Trimethylamine-N-oxide via Regulating the Gut Microbiota in Angiotensin II-Induced Hypertensive Mice

“…To investigate the microbiota community composition after BBR treatment, bacterial 16S rDNA gene sequencing assay was performed as previously described ( Shi et al, 2021 ). Briefly, fresh mice feces were collected and quickly frozen in liquid nitrogen and then stored at −80°C.…”