Osbelt, L. | Search bioRxiv

bioRxiv2023

An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition between Klebsiella species

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. The system allowed efficient and precise editing via enhanced lambda Red expression and functionally-validated complementation with the use of universal bookmark targets in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. * ImportanceCas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, K. michiganensis and K. grimontii. We then adapted a recently developed protocol for functional complementation based on universal bookmark targets applicable to all tested species. In summary, species specific adaptation of state-of-the-art genetic tools allow efficient gene deletion and complementation in types strains as well as natural isolates of KoSC members to study microbial interactions.

microbiology↗

bioRxiv2022

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

BackgroundKlebsiella 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. MethodsWe 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. ResultsIn 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. ConclusionsThis 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.

microbiology↗