Microbiome-mediated colonization resistance to carbapenem-resistant Klebsiella pneumoniae in ICU patients

Abstract

Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes serious intensive care unit (ICU)-acquired infections, yet the mechanisms of microbiota-mediated colonization resistance remain unclear. We analyzed the gut microbiome and metabolic profiles of healthy individuals and ICU patients, distinguishing those with and without CRKP colonization. ICU patients showed distinct microbial communities compared to healthy controls, and CRKP-positive patients exhibited unique microbial and metabolic signatures. We demonstrated that a healthy gut microbiome is essential for providing resistance against CRKP colonization in antibiotic-perturbed mouse with fecal microbiota transplantation (FMT). Both in vitro and in vivo experiments revealed that Lactiplantibacillus plantarum and Bifidobacterium longum as significant contributors to the decolonization of CRKP. Furthermore, we showed that probiotic supplementation or FMT significantly improved CRKP colonization resistance. The findings highlight that a specific gut microbiome is essential for resisting CRKP colonization, and that targeted microbiome restoration may serve as a viable strategy to prevent CRKP colonization in ICU patients.

Fig. 1. Distinct gut microbiome characteristics are observed among healthy individuals, carbapenem-resistant Klebsiella pneumoniae (CRKP) intestinal colonization-positive (CPRK-P), and CRKP intestinal colonization-negative patients (CRKP-N).

A–D Comparison of α-diversity in gut microbiota between healthy individuals (HCG) and ICU patients at admission (ICU-A): A Oberserved_species, p = 0.025; B Shannon index, p = 2.3e-0.8; C Simpson index, p = 4.3e-0.7; D Chao1 index, p = 0.03. E–H Comparison of α-diversity in gut microbiota between CRKP-P and CRKP-N groups: E Oberserved_species, p = 0.014; F Shannon index, p = 0.18; G Simpson index, p = 0.28; H Chao1 index, p = 0.019. I–L Comparison of α-diversity in gut microbiota between healthy individuals and CRKP-P groups: I Oberserved_species, p = 0.25; J Shannon index, p = 2.7e-0.7; K Simpson index, p = 4.6e-0.7; L Chao1 index, p = 0.26. M Beta diversity analysis between healthy individuals and ICU patients. N Beta diversity analysis between CRKP-P and their samples taken upon ICU admission (CRKP-PA). O Beta diversity between CRKP-negative and CRKP-positive patients upon ICU admission (CRKP-PA v.s. CRKP-NA). P Beta diversity between CRKP-N before and upon ICU admission (CRKP-NA v.s. CRKP-N). The Kruskal–Wallis test was used for significance testing.

Fig. 2. Lactobacillus and Bifidobacterium represent differential taxonomies in the gut microbiome of healthy individuals, Carbapenem-resistant Klebsiella pneumoniae (CRKP) intestinal colonization-positive ICU patients, and CRKP intestinal colonization-negative ICU patients.

A The gut microbiome of healthy individuals (HCG) and ICU patients (ICU-A) displays difference in the composition of gut microbiome at genus level. B The gut microbiome of CRKP-negative patients (CRKP-N) and their admission samples (CRKP-NA), CRKP-positive patients (CRKP-P), and their corresponding baseline samples upon admission (CRKP-PA) at the genus level. C–F Lefse analysis identified differential genera between HCG and ICU-A (C), CRKP-P and CRKP-N (D), CRKP-P and CRKP-PA (E), and CRKP-PA vs. CRKP-NA (F). G Sankey diagrams provided a more intuitive visualization, showing significant differences in Klebsiella between CRKP-PA and CRKP-P, while no significant changes were observed between CRKP-NA and CRKP-N. H The abundance of Bifidobacterium longum significantly decreased in CRKP-P compared with CRKP-PA. I Lactiplantibacillus plantarum abundance was significantly decreased in the CRKP-P compared with the CRKP-N. The Kruskal–Wallis test was used for significance testing.

Fig. 3. Carbapenem-resistant Klebsiella pneumoniae (CRKP) colonization alters gut microbiota function as predicted by PICRUSt2.

A–D CRKP colonization led to significant changes in the gut microbiota function of ICU patients. Bray–Curtis distance matrix based PCoA and PERMANOVA analysis indicated significant differences between CRKP-negative patients (CRKP-N) and CRKP-positive patients (CRKP-P) (A), CRKP-positive patients (CRKP-P) and their corresponding baseline samples upon admission (CRKP-PA) (B), CRKP-NA and CRKP-PA (C), while no significant difference was observed between CRKP-NA and CRKP-N (D). E KEGG level 2 pathway enrichment analysis showed a significant and contrasting trend in functional pathways between CRKP-P and the other sample groups. The Kruskal–Wallis test was used for significance testing.

Fig. 4. Untargeted metabolomics revealed significant changes in the gut metabolic profile of ICU patients following carbapenem-resistant Klebsiella pneumoniae (CRKP) intestinal colonization.

A OPLS-DA analysis indicated significant differences in the untargeted metabolomic profiles between healthy individuals and ICU patients. B The top 25 enriched metabolites displayed in panel (B). C A random forest algorithm identified the top 20 most important differential metabolites. Similarly, significant differences were observed between CRKP-negative patients (CRKP-N) and CRKP-positive patients (CRKP-P) (D–F), CRKP-positive patients (CRKP-P) and their corresponding baseline samples upon admission (CRKP-PA) (G–I), as well as CRKP-negative patients (CRKP-N) and their admission samples (CRKP-NA) (J–L).

Fig. 5. L. plantarum 21790 and B. longum 6188 effectively inhibit carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro and in vivo.

A Experimental design for CRKP in different concentrations of supernatant of probiotics in vitro. L. plantarum 21790 cultured with DeMan, Rogosa and Sharpe (MRS) medium, and B. longum 6188 cultured with Reinforced Clostridium Medium (RCM). B, C The inhibitory effects of supernatant from L. plantarum 21790 culture mediums on two clinical CRKP isolates (K. pneumoniae 020120 (B) and 020003 (C) demonstrated a significant concentration-dependent response. D, E The inhibitory effects of supernatant from B. longum 6188 culture mediums on two clinical CRKP isolates (K. pneumoniae 020120 (D) and 020003 (E)) demonstrated a significant concentration-dependent response. F Experimental design for the co-culture of probiotics and CRKP in filtered supernatant of healthy stool. An ex vitro model using a healthy donor gut microbiome (BLK) was employed. K. pneumoniae 020003 was introduced into this system (CON), alongside the supplement of L. plantarum 21790 (LPCO), B. longum 6188 (BLCO), or both strains concurrently (COPR). G Both L. plantarum 21790 and B. longum 6188 exert significant inhibitory effects on CRKP. H Experimental design for effects of probiotics on decolonization of CRKP in vivo. K. pneumoniae 020003 into mice after 1 week of treatment with meropenem (MEM) in water, and mice were supplemented with PBS (Ctl), 1.0 × 10⁹ CFU/ml of L. plantarum 21790 (LP), or B. longum 6188 (BL). I–L The CRKP decolonization effects of L. plantarum 21790 (BL) and L. plantarum 21790 (LP) on days 12 (I), 14 (J), 18 (K), and 25 (L). A reusable two-factor analysis of variances was used for comparison between different concentration groups and the control group.

Fig. 6. The role of a healthy microbiome in carbapenem-resistant Klebsiella pneumoniae (CRKP) decolonization.

A Experimental design for effects of antibiotics on CRKP colonization in vivo. The abundance of CRKP in the gut of mice was monitored by comparing different antibiotic treatments. The posttreatment group (PstTret group, showed in blue color): Mice were first treated with PBS for 10 days, followed by meropenem (MEM) treatment for 23 days; The intermittent treatment group (InterTret group, showed in green color): Mice were treated with MEM for 3 days before introducing K. pneumoniae 020003, then MEM was removed until day 11, at which point MEM treatment was resupplied. The pretreatment group (PreTret group, showed in red color): Mice were treated with MEM for 3 days before introducing K. pneumoniae 020003, with MEM treatment continuing until day 11, after which MEM was removed. The abundance of CRKP in mouse feces was measured daily. B CRKP abundance in feces in different groups during days 5–33. C Experimental design for effects of fecal transplantation (FMT) on CRKP decolonization in vivo. Control group (Ctl, showed in grayish blue): Mice was feeded to provide healthy fecal samples for FMT group during experiment. Phosphate Buffered Saline (PBS group, showed in purple): Mice were first treated with MEM for 14 days, 200 ul PBS was gavaged during days 8–28 as negative control. FMT group (showed in pink): Mice were first treated with MEM for 14 days, 200 ul FMT from the Ctl group was gavaged during days 8–28. About 200 μL of 1.0 × 10⁵ CFU/ml K. pneumoniae 020003 was gavaged on day 8. D CRKP abundance in feces in different groups during days 9–28.

Fig. 7. The ICU patient’s recruitment flow chart.

ICU intensive care unit, CRKP carbapenem-resistant Klebsiella pneumoniae, BMI body mass index.