Within-host evolution of Enterococcus faecium during longitudinal carriage and transition to bloodstream infection in immunocompromised patients

Abstract

Background: Enterococcus faecium is a leading cause of hospital-acquired infection, particularly in the immunocompromised. Here, we use whole genome sequencing of E. faecium to study within-host evolution and the transition from gut carriage to invasive disease.

Methods: We isolated and sequenced 180 E. faecium from four immunocompromised patients who developed bloodstream infection during longitudinal surveillance of E. faecium in stool and their immediate environment.

Results: A phylogenetic tree based on single nucleotide polymorphisms (SNPs) in the core genome of the 180 isolates demonstrated several distinct clones. This was highly concordant with the population structure inferred by Bayesian methods, which contained four main BAPS (Bayesian Analysis of Population Structure) groups. The majority of isolates from each patient resided in a single group, but all four patients also carried minority populations in stool from multiple phylogenetic groups. Bloodstream isolates from each case belonged to a single BAPS group, which differed in all four patients. Analysis of 87 isolates (56 from blood) belonging to a single BAPS group that were cultured from the same patient over 54 days identified 30 SNPs in the core genome (nine intergenic, 13 non-synonymous, eight synonymous), and 250 accessory genes that were variably present. Comparison of these genetic variants in blood isolates versus those from stool or environment did not identify any variants associated with bloodstream infection. The substitution rate for these isolates was estimated to be 128 (95% confidence interval 79.82 181.77) mutations per genome per year, more than ten times higher than previous estimates for E. faecium. Within-patient variation in vancomycin resistance associated with vanA was common and could be explained by plasmid loss, or less often by transposon loss.

Conclusions: These findings demonstrate the diversity of E. faecium carriage by individual patients and significant within-host diversity of E. faecium, but do not provide evidence for adaptive genetic variation associated with invasion.

Keywords: Enterococcus faecium; Genome sequencing; Within-host evolution.

Fig. 1

a Neighbour-joining tree of 180 E. faecium genomes used in this study. BAPS clusters 1, 2, 3 and 4 are inferred BAPS groups that all contained invasive isolates. The pie charts for BAPS groups 1, 2, 3 and 4 contain 13, 87, 26 and 26 isolates, respectively. The BAPS groups were on average 4797.97 SNPs apart. The average pairwise SNP distance for isolates within BAPS1, 2, 3 and 4 were 3.97, 2.13, 2.86 and 1.30, respectively. The BAPS run columns correspond to clustering results with five and ten values for the estimated numbers of clusters in the hierBAPS analysis. Each colour signifies one group. BAPS groups 1, 2, 3 and 4, which contained invasive isolates, were inferred with both parameter sets. The root of the tree is the midpoint of the two most distant taxa in the collection. Frequency of isolates based on patient (b) and source of isolation (c) across four BAPS groups

Fig. 2

Bayesian phylogenetic tree for patient B/BAPS2 group. Branches and nodes are coloured based on the inferred origin of ancestral strains. The size of the diamond signs shows the posterior probability values for the inferred status. The bars on the nodes denote 95% confidence intervals, i.e. the credible set that contains 95% of the sampled values

Fig. 3

Transmission tree of BAPS2 isolates from three patients reconstructed based on genetic distance and isolate dates. The edge numbers denote the number of mutations. Arrows model potential transmissions from ancestors

Fig. 4

Phylogenetic distribution of genes encoding antibiotic resistance and virulence factors. Note that only virulence factors that were variably present in the population are shown, meaning that some of the well-known virulence factors, e.g. sagA and atlAEfm, present in every isolate are not shown. The sequence file for the virulence factors studied here is provided in Additional file 1. Note that the vanB-containing isolate was phenotypically susceptible to vancomycin due to lack of vanR _B and vanS _B

Fig. 5

Frequency of resistant and susceptible isolates across the phylogenetic tree for antibiotics tested in this study