Colonizing multidrug-resistant bacteria and the longitudinal evolution of the intestinal microbiome after liver transplantation

Abstract

Infections by multidrug-resistant bacteria (MDRB) remain a leading cause of morbidity and mortality after liver transplantation (LT). Gut dysbiosis characteristic of end-stage liver disease may predispose patients to intestinal MDRB colonization and infection, in turn exacerbating dysbiosis. However, relationships between MDRB colonization and dysbiosis after LT remain unclear. We prospectively recruited 177 adult patients undergoing LT at a single tertiary care center. 16 S V3-V4 rRNA sequencing was performed on 723 fecal samples collected pre-LT and periodically until one-year post-LT to test whether MDRB colonization was associated with decreased microbiome diversity. In multivariate linear mixed-effect models, MDRB colonization predicts reduced Shannon α-diversity, after controlling for underlying liver disease, antibiotic exposures, and clinical complications. Importantly, pre-LT microbial markers predict subsequent colonization by MDRB. Our results suggest MDRB colonization as a major, previously unrecognized, marker of persistent dysbiosis. Therapeutic approaches accounting for microbial and clinical factors are needed to address post-transplant microbiome health.

Fig. 1

Significant clustering of samples collected during colonization by multidrug-resistant bacteria (MDRB). Nonmetric multidimensional scaling (NMDS) plots were constructed by using UniFrac β-diversity. Each point represents a single sample in the full data set (n = 703). The difference in microbiome community structure was compared during versus not during colonization by (a) any MDRB (dark yellow; colonized n = 254 vs. noncolonized n = 449), b vancomycin-resistant enterococci (VRE, dark pink; colonized n = 142 vs. noncolonized n = 561), c Enterobacteriaceae resistant to third-generation cephalosporins (Ceph-RE, dark green; colonized n = 162 vs. noncolonized n = 541), and d carbapenem-resistant Enterobacteriaceae (CRE, dark purple; colonized n = 46 vs. noncolonized n = 657) as identified by using culture-based methods (see Methods, Supplementary Methods). PERMANOVA pseudo-F-statistic and P-value are shown. Source data are provided as a Source Data file

Fig. 2

Pre-transplant α-diversity and β-diversity associated with liver disease etiology and severity. a Shannon and Chao α-diversity and b UniFrac β-diversity indices stratified by primary indication for LT (AIH n = 7; ARLD n = 7; HBV n = 8; HCV n = 37; NAFLD n = 14). c, d α-diversity and UniFrac β-diversity stratified by high vs. low MELD score at the time of LT (> 18 (n = 43) vs. ≤ 18 (n = 40)). e, f α-diversity and UniFrac β-diversity stratified by CTP class at the time of LT (a (n = 16) vs. b (n = 24) vs. c (n = 43)). a, c, e α-Diversity boxplots reflect median (horizontal center line), 25th and 75th percentile values (bottom and top bounds of boxes), and ranges (bottom and top of whiskers) for each category. Each panel shows P-values for univariate linear regression (see Table 2) as follows: ⁺ P < 0.1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. b, d, f PERMANOVA P-values and pseudo-F-statistic values calculated using UniFrac β-diversity distances are shown. LT liver transplant, AIH autoimmune hepatitis, ARLD alcohol-related liver disease, BILIARY biliary-related etiologies, HBV hepatitis B virus, HCV hepatitis C virus, NAFLD nonalcoholic fatty liver disease, PCLD polycystic liver/kidney disease, MELD model for end-stage liver disease, CTP Child–Turcotte–Pugh. Source data are provided as a Source Data file

Fig. 3

Progression of gut microbiome diversity before and after transplantation stratified by the underlying liver disease. Shannon (a) and Chao (b) α-diversity and UniFrac β-diversity (c) throughout the study period are shown. a, b AIH (13 patients, 45 samples), ARLD (19 patients, 69 samples), BILIARY (18 patients, 58 samples), HBV (10 patients, 45 samples), HCV (71 patients, 304 samples), and NAFLD (30 patients, 115 samples) were the most represented indications for LT in our cohort. These panels show Shannon (a) and Chao (b) α-diversity within each sample by the number of days post-LT of sampling for each disease group. Constrained linear mixed-effect (CLME) analysis was used to determine a global P-value describing the temporal trend in α-diversity within each disease group (see Supplementary Data 9 and Supplementary Data 10). c Changes in UniFrac β-diversity in the pre-, peri- (weeks 1–3), and early (months 1–3) and late (months 6–12) post-transplant periods are shown for patients with AIH (pre-LT n = 7; peri-LT n = 17; early post-LT n = 10; late post-LT n = 11), ARLD (pre-LT n = 7; peri-LT n = 20; early post-LT n = 20; late post-LT n = 22), BILIARY (pre-LT n = 4; peri-LT n = 17; early post-LT n = 12; late post-LT n = 25), HBV (pre-LT n = 8; peri-LT n = 11; early post-LT n = 11; late post-LT n = 15), HCV (pre-LT n = 37; peri-LT n = 88; early post-LT n = 89; late post-LT n = 90), and NAFLD (pre-LT n = 14; peri-LT n = 38; early post-LT n = 31; late post-LT n = 32). UniFrac β-diversity calculation and principal coordinates analysis (PCoA) were performed with the full dataset, and samples from each disease population were subsequently separated for visualization of temporal trends. Across all samples, the PCoA axis 1 explained 14.5% of observed variance, and PCoA axis 2 explained 5.6% of observed variance. LT liver transplant, AIH autoimmune hepatitis, ARLD alcohol-related liver disease, BILIARY biliary-related etiologies, HBV hepatitis B virus, HCV hepatitis C virus, NAFLD nonalcoholic fatty liver disease. Source data are provided as a Source Data file

Fig. 4

Microbial and clinical predictors of gut microbiome diversity across pre- and post-transplant phases. (Top) Top panel shows average Shannon α-diversity at each sampling time point within each disease category. (Left) Colonization by CRE or VRE, exposure to glyco-/lipo-proteins (i.e., vancomycin, daptomycin) within 14 days, diagnosis of ARLD, and high CTP score were independent predictors of decreased pre-transplant Shannon α-diversity (multivariate linear regression P < 0.0001). (Center) Peri-LT Shannon α-diversity was significantly lower given Ceph-RE colonization, exposure to group 2 β-lactams (e.g., piperacillin–tazobactam) or fluoroquinolones, high CTP score, peri-operative antibiotic regimen adjustments, and post-operative bleeding within 7 days of transplant (multivariate linear mixed-effect regression P < 0.0001). (Right) In the early post-LT period (months 1–3), CRE colonization was an independent predictor of Shannon α-diversity after adjusting for exposures to group 2 β-lactams, fluoroquinolones, and/or glyco-/lipo-proteins, high MELD, peri-operative antibiotic adjustment, and bile leak within 1 year post-LT (multivariate linear mixed-effect regression P < 0.0001). (Right) By the late post-LT phase (months 6–12), VRE colonization, group 2 β-lactam exposure, high MELD, peri-operative antibiotic adjustment, and bile leak or biliary stricture within 1 year post -LT were all independent predictors of Shannon α-diversity (multivariate linear mixed-effect regression P < 0.0001). LT liver transplant, AIH autoimmune hepatitis, ARLD alcohol-related liver disease, BILIARY biliary-related etiologies, HBV hepatitis B virus, HCV hepatitis C virus, NAFLD nonalcoholic fatty liver disease, MDRB multidrug-resistant bacteria, CRE carbapenem-resistant Enterobacteriaceae, VRE vancomycin-resistant enterococci, Ceph-RE Enterobacteriaceae resistant to third-generation cephalosporins, CTP Child–Turcotte–Pugh score, MELD model for end-stage liver disease. Source data are provided as a Source Data file