315. Modeling Strain-Dependent Variability in Carbapenem-Resistant Klebsiella pneumoniae Colonization in the Malnourished Host
Authors: Holowka Thomas , Xiao Jamie , Marimuthu Kalisvar , Tek Ng Oon , van Duin David , Bartelt Luther A 
Published: January 2025

Abstract

Background
Particular strains of Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) have achieved global dominance extending to developing countries where malnutrition is a potential risk factor for infection. Bacteria and host properties that determine colonization dynamics in this context are not understood. We hypothesized that strain variability in host cell adhesion and nutrient-utilization in vitro predicts in vivo intestinal colonization by CR-Kp in malnourished hosts.Figure 1:Strain-dependent cell adhesion by carbapenem-resistant Klebsiella pneumoniae(A) Adhesion to Colo-201 cells and/or (B) Caco-2 cells as percentage of bacteria recovered after co-culturing then washing off non-adherent cells. Bacterial quantification performed by culturing on MacConkey agar + Meropenem. Strains of interest indicated with arrows and colored-bars. All experiments showing results of 3 replicates.

Methods
Patient-derived isolates of CR-Kp were subject to in vitro assays testing adhesion to human colon cell lines. Select isolates were further tested for in vitro growth in minimal media with limiting protein availability. These isolates were additionally inoculated via orogastric lavage into C57BL/6 mice (n=6 per group) on a nutrient replete control diet (CD) or isocaloric protein-deficient diet (PD) and intestinal colonization was quantified with selective culture of feces and intestinal specimens on MacConkey agar containing meropenem.Figure 2:Nutrient limited growth of carbapenem-resistant Klebsiella pneumoniaeGrowth in vitro determined by serial absorption measurement of bacteria culture under different nutrient conditions. M9 minimal medium (M9mm; M9 solution +0.4% glucose) was used with or without casein peptone (0.16% or 0.016%) as a protein/amino acid source.

Results
Across &gt;100 screened human isolates of CR-Kp, adhesion to human colon cell lines ranged from &lt; 1% to 30% (Figure 1). Three high-adhesion isolates (Kp175, Kp4493 and Kp4605) and one low-adhesion isolate (Kp4404) were selected for further investigation (Table 1). All three high-adhesion isolates grew to varying degrees in protein-limited conditions, while Kp4404 required a protein source for growth (Figure 2). Mice inoculated with CR-Kp were stably colonized only if they were fed a PD rather than CD diet. Kp175 and Kp4605 demonstrated the highest fecal shedding and intestinal burden (102-104 CFU per mg), which was significantly greater on a PD versus CD diet (p&lt; 0.05 by Mann-Whitney test). In contrast, Kp4493 and Kp4404 inconsistently colonized mice regardless of diet (Figure 3).Figure 3:Diet-dependent intestinal colonization by carbapenem-resistant Klebsiella pneumoniae(A) 3-4 wk old C57BL/6 male mice initiated on nutrient-replete control diet (CD) or isocaloric protein-deficient diet (PD) and inoculated via orogastric lavage with 10^6 CFU of carbapenem-resistant Klebsiella pneumoniae (CR-Kp). (B) Weight measured over time along with bacterial burden in (C) fecal pellets and (D) intestinal tissue by culturing on MacConkey agar + Meropenem. 6 animals used per condition. * = p&lt;0.05 by Mann Whitney Test.

Conclusion
Our findings suggest a wide variability in intestinal cell adhesion and nutrient utilization by clinical isolates of CR-Kp. These in vitro properties incompletely predict in vivo colonization in a mouse model. Notably, host dietary deficiency promotes intestinal colonization by certain nutritionally resilient CR-Kp isolates, with potential implications for the global spread of high-risk strains in malnourished populations.Table 1:Genotypic, clinical and phenotypic characteristics of carbapenem-resistant Klebsiella pneumoniae
Adhesion percentage express as mean (standard deviation).

Disclosures
David van Duin, MD, PhD, Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support