Soutenance de thèse : Genome-wide identification of plasma resistance mechanisms in Klebsiella pneumoniae : Paving the way for the development of monoclonal antibodies against resistant bacteria

Par Aimé-Patrick Berwa (IBS/Groupe Pathogenèse bactérienne et Réponses Cellulaires)

The complement system is a crucial plasma component for innate host defence against bacterial infections and the main driver of K. pneumoniae (Kpn) elimination in human blood. However, Kpn has developed several ways to evade complement-mediated killing. Previous studies have shown that a persister-like subpopulation, exhibiting transient tolerance to complement, can survive plasma challenges in several Gram-negative bacterial species. This study aimed to explore the presence of plasma persisters in Kpn and to conduct transposon insertional mutagenesis (Tn-Seq) on a diverse set of clinical strains, with the goal of enhancing our understanding of plasma resistance mechanisms in this bacterial species.

We evaluated the plasma susceptibility of 28 diverse Kpn strains, including 20 ESBL-producing clinical isolates, over a 3-hour incubation period in native human plasma (NHP). These strains represented 19 different sequence types and carried 19 different K antigens and six O antigens. Only one strain was fully sensitive to NHP, while eight strains (28.6%) were resistant with mean survival rates between 10% and over 500%. Nineteen strains (67.8%) showed mean survival rates ranging from 0.008% to 8.8%, suggesting the presence of plasma persisters. Killing curves in plasma exhibiting a bi-phasic pattern confirmed the persistent phenotype. Eight diverse clinical strains were selected for whole-genome Tn-Seq experiments using the Himar1 mariner Tn, which stochastically inserts at thymine-adenine sites.

Sequencing identified 244 genes critical for survival in NHP compared to heat-inactivated plasma. Overall, the main genes identified either affected the polysaccharide structure or other components of the bacterial surface (such as ABC transporters, type IV bacterial secretion systems, or two-component systems), energy metabolism, iron import, and global regulation. In particular, 10 of the 12 genes in the enterobacterial common antigen (ECA) operon were called as hits, along with genes involved in various stages of lipid A or LPS core biosynthesis and modification (galE, lpxM, kdsC, and waaQ, as well as genes of the Mla system and the arnBCADTEF operon). Furthermore, a majority of resistant mutant strains isolated during Tn-Seq experiments exhibited significantly lower amounts of ATP following incubation in NHP, in comparison to the WT strains. Many Tn insertions were identified in several components of the bacterial respiratory chain (i.e. in the five genes cyoA, cyoB, cyoC, cyoD, and cyoE, that constitute the cytochrome ubiquinol oxidase operon, as well as in cydA, cydB, nuoF, ndh, nqrC, ubiI, and ubiJ), and in purine metabolism genes (purA, purH, and hpt). Among common pathways, the wzzE (ECA biosynthesis) and arnT (LPS synthesis) genes were found to be under-represented in six and five Tn libraries, respectively, while the ackA (amino acid synthesis), csrD (transcriptional regulator), cyoB (oxidative phosphorylation), and ndh (oxidative phosphorylation) genes were over-represented in five Tn libraries each. However many hits were strain-specific.

The response of K. pneumoniae to the bactericidal activity of plasma is complex. Multiple pathways, particularly those involved in the biosynthesis and architecture of bacterial surface components, are crucial for bacterial resilience against plasma and complement-mediated killing. In an era of rising antibiotic resistance and the ongoing search for novel therapeutic options, targeting these pathways may offer promising strategies to enhance or restore complement activity.