CC5 strains facilitate transfer of vancomycin resistance from co-infecting Enterococci.

A study to sequence all 12 known strains of hospital-acquired vancomycin-resistant Staphylococcus aureus (VRSA) in North America has confirmed that each strain evolved completely independently to the others. The sequencing project, carried out by an international Harvard Medical School-led team, has provided new insights into how the bacterium manages to acquire the vancomycin-resistance Tn1546 transposon from Enterococcus strains and why the resulting VRSA bacteria are so well-suited to co-existing with other infective bacteria in the same host.

Overall, the sequencing data indicated that all the North American VRSA strains last shared a common ancestor over 50 years ago, at about the same time that methicillin was introduced but some 40 years before vancomycin resistance was first observed.

Michael S. Gilmore, Ph.D., and colleagues report their findings in mBio in a paper titled “Comparative Genomics of Vancomycin-Resistant Staphylococcus aureus Strains and Their Positions within the Clade Most Commonly Associated with Methicillin-Resistant S. aureus Hospital-Acquired  Infection in the United States.”

Since 2002 there have been 12 cases of VRSA infection in the U.S., all of which have been caused by bacteria of the clonal cluster 5 (CC5) clade. All are believed to have evolved when methicillin-resistant Staphylococcus aureus (MRSA) acquired the vancomycin resistant transposon through horizontal transmission from an Enterococcus bacterium during the course of an infection.

As the researchers explain, “such transfer requires that donors and recipients co-exist intimately in a mixed community and that they achieve a population size that allows them to overcome inefficiencies and obstacles to transfer, genetic element establishment, and resistance expression.”

To look for traits that may render the CC5 clade more capable of acquiring vancomycin resistance and to determine a basic evolutionary tree, the team sequenced the 12 known VRSA strains and generated a core gene sequence-based phylogeny based on nearly 2,000 genes that are present in all CC5 S.aureus genomes.

An initial comparison of Tn1546 sequences indicated that the vancomycin resistance transposon had been acquired by each VRSA strain independently and hadn’t just been acquired by one strain and subsequently passed down vertically. In other words, each strain evolved independently. Moreover, in some VRSA strains the Tn1546 sequence had been inserted into a Staphylococcus plasmid, whereas other strains retained large enterococcal plasmids containing the Tn1546 sequence. In two of the strains, isolated from the same patient, Tn1546 was found to reside on a plasmid generated by the fusion of an enterococcal and an S. aureus plasmid.

Significantly, none of the VRSA strains exhibited identifiable changes to either the Sau1 or type III-like restriction systems that normally prevent barriers to foreign DNA entry. This indicated that rather than removing the natural block to the transmission of DNA, the VRSA strains had acquired within the νSaβ island.

Notably, the North American VRSA strains all lack the bsa operon  normally located within νSaβ, which encodes a lantibiotic bacteriocin that is active against other Gram-positive bacterial species. Lack of bsa essentially facilitates the co-existence of other bacteria alongside CC5 S.aureus in the same infected patient, increasing at least the opportunity for horizontal transmission of genetic elements including Tn1546 from enterococcal strains.

And in place of the bsa operon, the VRSA bacteria all harbored a unique set of genes for enterotoxins that attack the host and thus act to aid survival of both S.aureus strains and other bacteria invading the infected site. Both these features would support co-existence of different bacterial species. Nearly all the VRSA strains exhibited much larger set of lipoprotein genes than non-CC5 strains, which could act to hamper natural host immune responses.

All the VRSA strains also exhibited mutations in dpra, which in other bacteria is known to influence how foreign DNA is assimilated. Such mutations would potentially aid the successful establishment of transferred genetic elements including Tn1546.

Interestingly, the authors note, absence of the bacteriocin operon and presence of a nearly identical complement of superantigens has also been found in epidemic MRSA strains found in the U.K., even though these strains have very different genetic backgrounds. “This suggests active selection for this version of νSaβ in the hospital environment,” they write.

 “VRSA (and other CC5) strains were found to possess a constellation of traits that appears to be optimized for proliferation in precisely the types of polymicrobic infection where transfer could occur,” the researchers conclude. “They lack a bacteriocin operon that would be predicted to limit the occurrence of non-CC5 strains in mixed infection and harbor a cluster of unique superantigens and lipoproteins to confound host immunity. A frameshift in dprA, which in other microbes influences uptake of foreign DNA, may also make this lineage conducive to foreign DNA acquisition.”

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