Phages against non-capsulatedKlebsiella pneumoniae: broader host range, slower resistance

Abstract

Background: Klebsiella pneumoniae (Kp) is an ecologically generalist bacterium but also an opportunistic pathogen responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, few advances have been made in the use of virulent phages as alternative or complement to antibiotics to treat Kp infections. The efficiency of phages relies on their ability to recognize and attach to the bacterial surface structure, and in the case of Kp, capsule (K) is the main surface structure. However, Kp capsule is highly polymorphic and the majority of classically isolated phages are specific for unique K-types, limiting therapy prospects. In this study, we demonstrate the feasibility of an innovative strategy consisting in isolating phages that target capsule-deficient mutant Kp strains, and compare such phages with anti-capsulated cells phages phylogenetically and through in vitro and in vivo experiments. Methods: We isolated 27 phages using 7 capsule-deficient Kp strains as hosts (anti-Kd phages), and 41 phages against 7 wild-type (wt) Kp strains (anti-K phages). We evaluated and compared phenotypically and genotypically their host range, resistance emergence and selected mutations and in-vivo activity. Results: In vitro, anti-Kd phages showed a broader host-range, with most phages being able to infect non-capsulated mutants of multiple sublineages and O-antigen locus types. Besides, the emergence of bacterial subpopulations non-susceptible to anti-Kd phages was slower when compared to anti-K phages and with a different range of genomic differences. One anti-Kd phage (mtp5) was shown to infect non-capsulated Kp strains belonging to 10 of the 12 known O-antigen types. Moreover, this phage was able to replicate in the gut of mice colonised with the wt (capsulated) parent strain. Conclusions: This work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of narrow host-range of anti-K phages. Anti Kd-phages may be active in infection sites where capsule expression is intermittent or repressed, or in combination with anti-K phages, which often induce loss of capsule escape mutants.

Genetic determinants of host tropism in Klebsiella phages

“…It can also be speculated that S8/S9 phages might preferentially encounter receptors in the equator of the cell surface, where the capsule is thinner, 66 or in bacteria with lower capsule production levels. 40 , 67 …”

“… 39 Additionally, some phages without Dpos have been described, but their determinants of host-range remain elusive. 20 , 35 , 40 …”

A panel of diverse Klebsiella pneumoniae clinical isolates for research and development

“…However, in addition to testing antibiotics on strains with diverse AMR gene content, strain diversity is critical for assessing the efficacy of many emerging therapeutics, including phage therapy, vaccines and capsule polysaccharide-targeted approaches [47–49]. The main structural receptor for anti- Klebsiella phages is the external capsular polysaccharide, although recent work suggests that phages also attach to other outer-membrane structures below the capsule, including the O-antigen [50, 51]. The panel described herein represents 54 of the 77 distinct capsule types identified by serological methods [52] and 8 predicted O serotypes [38, 53], providing a robust representation of outer-membrane protein diversity to test anti- Klebsiella phages.…”

A Panel of Diverse Klebsiella pneumoniae Clinical Isolates for Research and Development

“…The copyright holder for this preprint this version posted August 18, 2022. ; https://doi.org/10.1101/2022.08.17.504361 doi: bioRxiv preprint emerging therapeutics including phage therapy, vaccines, and capsule polysaccharides targeted approaches (38)(39)(40). The main structural receptor for anti-Klebsiella phages is the external capsular polysaccharide however recent work suggests that phages also attach to other outer membrane structures below the capsule, including the O-antigen (41,42). The panel described herein represents 54 of the 77 distinct capsule types identified by serological methods (43) and 8 predicted O serotypes (27,44), providing a robust representation of outer membrane protein diversity to test anti-Klebsiella phages.…”