The Gram-positive bacterium Enterococcus faecium is a member of the ESKAPE pathogens for which drug resistance has been a growing problem. How E. faecium becomes drug resistant has been a long-standing question, and is the focus of a new study now available in Antimicrobial Agents and Chemotherapy. A research team led by senior scientist Louis Rice has identified chromosomal regions where homologous recombination facilitates incorporation of genes conferring beta-lactam and vancomycin resistance.
Enterococci have been problematic for many decades, particularly in the context of health care-associated infections. Many Enterococcal species have intrinsic resistance to various drugs, meaning that they are unaffected by the mechanism by which certain drugs act. Typing infections to the species level is important in clinical diagnoses to determine whether a species is inherently sensitive or resistant to a particular compound. However, Enterococci are also masters of sharing DNA, including drug-resistance genes, meaning that many infections have acquired drug resistance as well. In their study, the researchers investigated the incorporation of genetic resistant determinants in E. faecium.
For many years, most Enterococcal infections were caused by E. faecalis, but cases of E. faecium disease have been steadily increasing. “This is a serious problem, because E. faecium is far more difficult to treat,” says Monica Garcia-Solache, first author of the AACJournal report. Almost all E. faecium strains are intrinsically resistant to penicillins and some strains acquire high-level resistance through mutations in key proteins involved in cell wall building, explains Garcia-Solache. Enterococci are intrinsically resistant to aminoglycosides and clindamycin, and are frequently resistant to erythromycin and the fluoroquinolones. Particularly troubling is that 77% of all U.S. E. faecium clinical isolates are resistant to vancomycin, compared to only 9% vancomycin resistance in E. faecalis. “These high resistance levels make treatment difficult and limited to a small range of useful antibiotics,” she says. “Learning the mechanisms of resistance acquisition in E. faecium may lead to better therapeutic approaches or novel targets to treat it.”
The researchers investigated the transfer of genetic determinants for drug resistance, concentrating on the penicillin binding protein 5 (pbp5) operon and the vancomycin resistance cassette (vanB) from a clinical isolate. Pbp5 is a gene that encodes a transmembrane protein important for cell wall assembly and is found in all wild-type E. faecium strains. The vanB element is a complex cassette that encodes different proteins that modify the peptidoglycan precursors that are incorporated into the cell wall, effectively altering the vancomycin target in a way that renders the antibiotic ineffective. The pbp5 gene is chromosomally encoded, while vancomycin resistance is normally encoded by transposons that can be plasmid or chromosomally borne.
How is resistance to penicillins and vancomycin transferred? To find out, the researchers used two E. faecium strains: a donor strain that was vancomycin-resistant and highly resistant to penicillins and a recipient strain that was vancomycin-susceptible and less resistant to penicillins than the donor. The strains were then incubated together and transconjugants selected by their resistance profiles were further investigated by whole genome sequencing. This detailed information allowed the scientists to determine that transfer of pbp5 and the adjacent vanB operon was almost always linked, and that the strain acquiring vancomycin resistance replaced its own pbp5 allele with that of the donor strain through homologous recombination (see schematic, right).
This cotransfer didn’t rely on the transposon activity of the vanB element, however. The researchers observed pbp5 and vanB operon – as well as additional chromosomal DNA – was incorporated into the recipient chromosome. A large conjugative plasmid present in the donor strain was frequently – but not always – transferred, and may be initiating the DNA mobilization from one strain to the other. “This suggests there is more than one mechanism to mobilize DNA from one strain to another without the need of conventional transposable elements,” says Garcia-Solache. Further, “our findings suggest that virtually any part of the genome can be transferred and replaced, given the right selection pressure.”
Discovering the involvement of multiple mechanisms in E. faecium sharing or incorporating new DNA emphasizes the importance of basic genetic research. This is a successful pathogen in health care settings in part due to its ability to resist antibiotic action. As researchers look for ways to combat the spread of antibiotic resistance, one means is to inhibit the spread of resistance genes between bacteria. But when it comes to E. faecium resistance and genetic swapping, “there is not a ‘one size fits all’ mechanism this bacterium uses,” says Garcia-Solache.
-- Julie Wolf