Short-read data for the 96 genomes sequenced in this study are available at the European Nucleotide Archive (ENA), accession number PRJNA344739. The long-read sequence dataset used for the assembly of the genome of strain A_020709_82 is available at ENA, accession number CP018128. †These authors contributed equally to this work Data statement: All supporting data, code and protocols have been provided within the article or through supplementary data files. One supplementary table and two supplementary figures are available with the online version of this article.

14 15 Conclusions 44 Our findings show that the E. faecium genome is highly dynamic during asymptomatic 45 colonization of the patient gut. We observe considerable genomic flexibility due to 46 frequent horizontal gene transfer and recombination, which can contribute to the 47 Results 105 106 Isolate collection and patient hospital stay 107 This study used vancomycin-and ampicillin-resistant E. faecium (VRE and ARE, 108 respectively) isolates that were collected and stored in the period 2001 -2008 as part 109 of routine diagnostics and infection prevention interventions at the University Medical 110Center Utrecht, the Netherlands (figure 1). Analysis of the collected isolates with 111 anonymized patient data, showed that for five patients multidrug-resistant E. faecium 112 isolates were collected over a period of >1 year. We sequenced the genomes of 96 113 isolates, all of which were determined to be ampicillin-resistant using a previously 114 described method [26]. Using Abricate [27], we found that 38 and 21 isolates carried 115 the vanA or vanB operon. Further information on the antibiotic resistance profiles of 116 the strains sequenced in this study is provided in Supplementary Table 1. The time 117 span between the first and the last isolate collected from a single patient ranges from 118 15 months (patient B) to 6.5 years (patient C). 119 120 Genetic diversity of E. faecium patient isolates 121To be able to place the collected isolates in the larger E. faecium population, we 122 created a SNP-based, recombination-filtered phylogenetic tree using the 96 genomes 123 sequenced in this study and 70 previously described E. faecium genome sequences 124 that represent the global E. faecium population [8]. This phylogenetic tree is based on 125 1448 core genes and a total of 77,909 SNPs. Out of the 96 patient isolates, 95 126 clustered into clade A1, a clade of hospital-associated E. faecium strains 127 (Supplementary figure 1). The remaining isolate clustered with strains that were 128 previously assigned to clade A2. 129 130The relatively large diversity of the 70 publicly available E. faecium genome 131 sequences, limited the resolution of the phylogenetic relationships between the patient 132

Enterococcus faecium is a nosocomial, multidrug-resistant pathogen. Whole genome sequence studies revealed that hospital-associated E. faecium isolates are clustered in a separate clade A1. Here, we investigated the distribution, integration site and function of a putative iol gene cluster that encodes for myo-inositol (MI) catabolism. This iol gene cluster was found as part of an ~20 kbp genetic element (iol element), integrated in ICEEfm1 close to its integrase gene in E. faecium isolate E1679. Among 1644 E. faecium isolates, ICEEfm1 was found in 789/1227 (64.3 %) clade A1 and 3/417 (0.7 %) non-clade A1 isolates. The iol element was present at a similar integration site in 180/792 (22.7 %) ICEEfm1-containing isolates. Examination of the phylogenetic tree revealed genetically closely related isolates that differed in presence/absence of ICEEfm1 and/or iol element, suggesting either independent acquisition or loss of both elements. E. faecium iol gene cluster containing isolates E1679 and E1504 were able to grow in minimal medium with only myo-inositol as carbon source, while the iolD-deficient mutant in E1504 (E1504∆iolD) lost this ability and an iol gene cluster negative recipient strain gained this ability after acquisition of ICEEfm1 by conjugation from donor strain E1679. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources. In an intestinal colonization mouse model the colonization ability of E1504∆iolD mutant was not affected relative to the wild-type E1504 strain. In conclusion, we describe and functionally characterise a gene cluster involved in MI catabolism that is associated with the ICEEfm1 island in hospital-associated E. faecium isolates. We were unable to show that this gene cluster provides a competitive advantage during gut colonisation in a mouse model. Therefore, to what extent this gene cluster contributes to the spread and ecological specialisation of ICEEfm1-carrying hospital-associated isolates remains to be investigated.