Functional characterization of an inositol gene cluster

Enterococcus faecium is one of the leading nosocomial multidrug-resistant pathogens globally. Whole genome sequence studies revealed that the hospital-associated E. faecium isolates are clustered in a separate clade, designated clade A1. Among clade A1 isolates, several virulence associated genes and genomic islands encoding gene clusters involved in carbohydrate metabolism were identified to be enriched and thought to be involved in gut colonization of patients and subsequent infections in especially immunocompromised patients. Here, we investigated the evolution, 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 integrated close to the integrase of ICEEfm1 in E. faecium isolate E1679. We determined the distribution of ICEEfm1 with and without the ~20 kbp genetic element among the previously generated sequence clusters (SC) based on a core gene alignment for 1,644 E. faecium isolates. ICEEfm1 was found in 789/1,227 (64.3%) clade A1 and 3/417 (0.7%) non-clade A1 isolates and the ~20 kbp genetic element in 180/792 (22.7%) ICEEfm1-containing isolates. Gene neighborhood analysis revealed for all isolates a similar integration site of the ~20 kbp genetic element in ICEEfm1. Examination of the phylogenetic tree revealed genetically closely related isolates but differences in presence/absence of ICEEfm1 and/or the ~20 kbp genetic element, suggesting either independent acquisition or loss of both elements.Two E. faecium isolates E1679 and E1504 that contained the iol gene cluster were able to grow on a minimal medium with only myo-inositol as a carbon source (M1-MI), while the iolD-deficient mutant (E1504?iolD) lost the ability to grow on MI-M1. In contrast, conjugation experiments showed that an iol gene cluster negative recipient strain that was not able to grow on M1-MI gained this ability after acquisition of ICEEfm1 including the iol gene cluster from the donor E1679 strain. Using an intestinal mouse colonization model we demonstrated that intestinal colonization of the E1504?iolD mutant was not affected relative to the wild-type E1504 strain. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources, which likely explains the outcome of the mouse colonization experiment.In conclusion, in this study we have shown that a large majority of hospital-adapted isolates either acquired or lost ICEEfm1 with and without the ~20 kbp genetic element including the iol gene cluster. In addition, the iol gene cluster-containing isolates are able to utilize MI as sole carbon source, but only in the absence of other carbon sources, which is not the case in their natural environment, the gut. How this adaptive mechanism provides isolates circulating in hospitals a selective advantage remains to be investigated.