Coexistence of virulence genes in methicillin-resistant Staphylococcus aureus clinical isolates.

INTRODUCTION
The pathogenic versatility of Staphylococcus aureus is attributed to various virulence genes, including enterotoxins and hemolysins.


METHODS
Here, the virulence genes in 177 nosocomial MRSA strains in Porto Alegre, Brazil were detected by PCR.


RESULTS
The overall prevalence rates were as follows: sea, 4.5%; pvl, 18.6%; tst, 27.7%; hla, 87.6%; and hld, 90.4%. No strain contained all tested genes. However, there was frequent coexistence of tst with pvl and hla with hld (40.7% and 26.6%, respectively).


CONCLUSIONS
Horizontal transfer of virulence genes is very common in S. aureus, as suggested by the frequent coexistence of several virulence genes.

Virulence and antimicrobial resistance are two characteristics that do not always coexist in bacteria, since both are associated with large metabolic burdens, resulting in decreased fitness 1 . However, if the microorganism harbors the genetic capacity allowing it to produce either the determinants of antimicrobial resistance or virulence factors in specific situations, if a host demonstrate weakness, then both might be concomitantly expressed. Staphylococcus aureus has several resistance mechanisms and several virulence genes that promote adhesion to host cells, tissue invasion, and escape from the immune system, to establish a chronic infection 2 .
Methicillin resistance is the most prevalent resistance acquired by S. aureus, and it is highly disseminated worldwide. As an opportunistic microorganism, S. aureus takes advantage of its existence as both a commensal and a pathogen to survive in the host, since selective pressures promote adaptation. Moreover, when living with other commensal bacteria, such as Staphylococcus epidermidis and Enterococcus, S. aureus may acquire additional resistance or virulence characteristics 1,3 . Because many of these genes are encoded on mobile genetic elements, such as plasmids or prophages, they can be transmitted between strains by horizontal transfer 4 . In addition to its antimicrobial resistance arsenal, there are several virulence genes that make S. aureus such a versatile pathogen. The most frequent virulence genes encode toxins such as enterotoxins, Panton-Valentine leukocidin, and toxic shock syndrome toxin, and hemolysins such as alpha-, beta-, and delta-hemolysins. The expression of these virulence factors is coordinated by quorumsensing activity, a cell-communication system that controls gene expression in response to population density 5,6 .
In this study, we report the coexistence of quorum-sensing regulated virulence genes among a collection of methicillinresistant Staphylococcus aureus (MRSA) strains obtained from hospitals in Porto Alegre, Brazil. Our findings provide insight into the pathogenesis and evolution of MRSA.
The overall prevalence rates of the virulence genes among the HA-MRSA isolates were as follows: sea: 4.5%; pvl: 18.6%; tst: 27.7%; hla: 87.6%; and hld: 90.4%. The distribution pattern is presented in Figure 1. The respiratory tract (40.7%) and blood (22.6%) were the major MRSA recovery sites, which were followed by the minor sites.
Some MRSA isolates (45%) recovered from blood cultures contained both hla and hld, and 92.5% that contained both genes, harbored another gene. In 10 isolates, no virulence gene was found, and no strain contained all genes.
Staphylococcus aureus is a dangerous and versatile pathogen that can cause a multitude of different diseases. Most frequently, it causes infections of the skin and respiratory tract 4 . S. aureus secretes a group of peptides that can damage the host cell plasma membrane, such as pore-forming toxins like hemolysins 9 . The vast majority of S. aureus strains harbors all four hemolysin genes, hla, hlb, hld, and hlg. It is notable that the two hemolysin genes we tested in this study showed the highest coexistence, highlighting their importance to virulence.
Toxic shock syndrome toxin-1(TSST-1) is a potent superantigen, and it is the most common cause of toxic shock syndrome. It is produced exclusively by S. aureus, and approximately 20% of natural isolates are TSST-1 producers 10 . In this study, 27.7% of the MRSA isolates harbored the tst gene, which is inserted into a pathogenic island 11 , which is widely assumed to be a mobile element. Therefore, TSST-1 can be transmitted through horizontal transfer, which is responsible for its spread among S. aureus strains.
The Panton-Valentine leucocidin (PVL) toxin in S. aureus is responsible for the destruction of polymorphonuclear and mononuclear cells, through necrosis and apoptosis, which usually causes skin or soft tissue infections and necrotizing pneumonia 12 . In our study, pvl was detected in 18.6% of the HA-MRSA strains, as was observed in previous reports 13,14 . The increasing prevalence of pvl in HA-MRSA is likely because of the presence of infective PVL phages 13 .
In summary, horizontal transfer of virulence genes is very common in S. aureus, which is suggested by the considerable coexistence of several virulence genes in this study.