Functional characterization of a gene cluster responsible for inositol catabolism associated with hospital-adapted isolates of Enterococcus faecium.

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.

Enterococcus faecium genome dynamics during long-term asymptomatic patient gut colonization.

Enterococcus faecium is a gut commensal of humans and animals. In addition, it has recently emerged as an important nosocomial pathogen through the acquisition of genetic elements that confer resistance to antibiotics and virulence. We performed a whole-genome sequencing-based study on 96 multidrug-resistant E. faecium strains that asymptomatically colonized five patients with the aim of describing the genome dynamics of this species. The patients were hospitalized on multiple occasions and isolates were collected over periods ranging from 15 months to 6.5 years. Ninety-five of the sequenced isolates belonged to E. faecium clade A1, which was previously determined to be responsible for the vast majority of clinical infections. The clade A1 strains clustered into six clonal groups of highly similar isolates, three of which consisted entirely of isolates from a single patient. We also found evidence of concurrent colonization of patients by multiple distinct lineages and transfer of strains between patients during hospitalization. We estimated the evolutionary rate of two clonal groups that each colonized single patients at 12.6 and 25.2 single-nucleotide polymorphisms (SNPs)/genome/year. A detailed analysis of the accessory genome of one of the clonal groups revealed considerable variation due to gene gain and loss events, including the chromosomal acquisition of a 37 kbp prophage and the loss of an element containing carbohydrate metabolism-related genes. We determined the presence and location of 12 different insertion sequence (IS) elements, with ISEfa5 showing a unique pattern of location in 24 of the 25 isolates, suggesting widespread ISEfa5 excision and insertion into the genome during gut colonization. Our findings show that the E. faecium genome is highly dynamic during asymptomatic colonization of the human gut. We observed considerable genomic flexibility due to frequent horizontal gene transfer and recombination, which can contribute to the generation of genetic diversity within the species and, ultimately, can contribute to its success as a nosocomial pathogen.