From MRSA nasopharyngeal carriage to infection
S. aureus has the capacity to both colonize human hosts without causing too much harm and act as a major pathogen. The role of nasopharyngeal carriage of S. aureus in the spread of microorganisms, including MDR strains, and as a risk factor for infection in the host is well documented .
Several genomes of S. aureus have now been completely sequenced. Sequence analysis has shown that all S. aureus strains contain major virulence factors and that they have the capacity to become invasive . Staphylococcal virulence is probably better explained by modulation of the expression of virulence factors by accessory genes. However, and most importantly, individual variations in intermittent or persistent carrier status cannot be understood without identification of host factors that contribute to colonization. The complete picture of the contribution of host genetic polymorphism to nasal carriage and the occurrence of infections is far from being completely elucidated. Recent studies have shown associations between polymorphisms in interleukin-4, complement factor H and C-reactive protein, on the one hand, and S. aureus carrier status and the occurrence of boils on the other . The observation that the host genotype was associated with carriage of certain staphylococcal types confirms that nasopharyngeal carriage results from complex interactions among host, pathogen, and environment, including antibiotic selective pressure.
Dissemination of MRSA: tracing the bacteria and identifying new reservoirs
The spread of MDR bacteria results from an interplay of transfer of resistance genes between strains, bacterial species or genera and of dissemination of bacterial clones in various environments. Gene transfer occurs through horizontal transfer of plasmids, transposition (e.g. vanA borne by transposon Tn1546 in enterococci), dissemination of resistance cassettes (e.g. SCCmec in MRSA), or transformation (e.g. pbp genes of Streptococcus pneumoniae), depending on the microorganism and on the genetic support of the resistance determinant.
Tools are available for tracing the sources of dissemination and deciphering the genomic evolution of MDR organisms that allow adaptation to diverse hosts and environments. Various genotyping techniques based on PCR (rep-PCR, amplified fragment length polymorphism), pulsed-field gel electrophoresis, sequencing (multilocus variant analysis, single-locus sequence typing, multilocus sequence typing (MLST)) and DNA microarrays may be used.
The choice of the technique will depend on the objectives of the study, which can be limited to local outbreaks or can extend to national or global surveillance; this has recently been discussed by van Belkum et al. .
In staphylococci, the mecA gene encoding penicillin-binding protein 2a, responsible for methicillin resistance, is borne by 21–67-kb mobile genetic elements, termed the staphylococcal cassette chromosome mec (SCCmec) . The SCCmec element integrates into the S. aureus chromosome at a unique site (attBSCC). SCCmec elements are characterized by the presence of flanking terminal direct and, in most cases, inverted repeats, two essential genetic components (the mec gene complex and the ccr gene complex), and three junkyard (or jigsaw) regions (J). To date, six classes (A, B, C1, C2, D, and E) of mec gene complex and five types (1, 2, 3, 4, and 5) of ccr gene complex have been described. Different combinations of ccr and mec complexes allowed the definition of six types in S. aureus, with subypes defined as variations in the J regions . SCCmec typing is used in combination with other typing techniques, such as MLST, for tracing MRSA clones. Using different typing techniques, in particular MLST, the spread of hospital-acquired (HA)-MRSA and community-acquired (CA)-MRSA clones has been traced. The role of travel and of population migrations in the spread of MDR organisms is now well established. For instance, in Belgium, 25% of CA-MRSA infections were acquired after travel abroad . The origin of new epidemic HA-MRSA clones is still under discussion. They may result from international cross-border spread or de novo emergence of clones by SCCmec type IV insertion into recipient methicillin-susceptible S. aureus lineages.
New reservoirs of MRSA have been identified using MLST techniques. In The Netherlands, unexpected cases of MRSA carriage and infection were linked to pig farming . Isolates belonged to sequence type ST398. This particular type was found in staphylococci from pigs in France , and was highly prevalent in MRSA isolated in 39% of pigs in slaughterhouses in The Netherlands .
Finally, these powerful typing techniques have shed light on the circulation, the reservoirs and the origin and evolution of MDR organisms. However, predictions about future epidemic MDR clones and evolution remain uncertain.
MRSA strains have abolished borders between the community and hospitals
As already mentioned, MRSA isolates have spread internationally. However, they have traditionally been associated with infections in hospitals, to which they were mostly confined. Recently, new MRSA strains have emerged and rapidly spread in the community. They cause infections that are acquired by persons who have not been recently hospitalized or undergone a medical procedure (related to, for example, surgery, catheters, percutaneous medical devices, or dialysis). These so-called CA-MRSA strains are responsible for infections with a particular clinical presentation, and are associated with skin and soft tissue infections, such as pimples and boils, that occur in previously healthy and young persons.
Although there is no consensus definition, CA-MRSA isolates from throughout the world have several common characteristics. The most important are the production of Panton–Valentine leukocidin (PVL), which is infrequent in other S. aureus strains, and the presence of short SCCmec elements (of type IV or V). CA-MRSA isolates initially lacked multiple resistance to antibiotics. However, they can be classified as MDR organisms, because they are resistant to the β-lactam class of antibiotics, which includes major antistaphylococcal agents, and to some other antimicrobials, such as tetracyclines and fusidic acid, when isolated in Europe. In addition, new variants of CA-MRSA clones resistant to clindamycin and quinolones have recently emerged .
In the USA, CA-MRSA became more prevalent than methicillin-susceptible S. aureus in community-acquired S. aureus infections. In a recent study including 422 emergency department patients, 59% of S. aureus isolates from skin and soft tissue infections requiring drainage were resistant to methicillin, with variations from 20% to 72%, depending upon the state . Most infections in the USA are due to an MRSA clone characterized by sequence type ST8 and called USA300.
Two recent major events in the evolution of the USA300 clone were observed. First, it started to infiltrate hospitals and to replace the traditional HA-MRSA strains. From 2000 to 2006, the proportion of MRSA strains isolated from hospital-onset bloodstream infections and displaying a community phenotype of resistance increased from 24% to 49% in a US hospital . The total number of bloodstream infections remained stable, suggesting that the CA-MRSA strains replaced the HA-MRSA strains without causing additional infections. However, spread of CA-MRSA infections in hospitals is worrying, as it would occur among a more debilitated, older patient population and would provoke more severe infections. In addition, the skin tropism may add to the capacity of CA-MRSA to disseminate, which may eventually lead to an increased global burden of infections.
Subsequently, variants of USA300 MRSA that were resistant to clindamycin, ciprofloxacin and mupirocin became common among men who have sex with men . Possibly, MDR MRSA infections will have to be added to the list of sexually transmitted diseases.
CA-MRSA strains have also been described on other continents. Clones were initially reported as being continent-specific, with the ST80 type spreading in Europe. However, recent data show intercontinental exchanges of CA-MRSA clones and emergence of new PVL-positive clones, resulting in a more complex situation.
The prevalence of CA-MRSA is not uniform in Europe. It ranges from low in France (3.6% of MRSA) and in England and Wales [15,16] to high in Greece, with 75% of MRSA strains in the community containing PVL genes . The prevalence is also high at the southern boundary of Europe, with 72% of MRSA strains containing PVL genes in Algeria . The high prevalence of CA-MRSA in certain countries was found to be associated with an increased prevalence of CA-MRSA in hospitals  and, in Algeria, with diversification of the resistance profiles .
Despite the recent developments, the European ST80 clone seems to have less potential for dissemination than the USA300 clone. The epidemiological success of the USA300 clone (which may be considered as a ‘superbug’) has tentatively been explained by several particular characteristics. USA300 produces novel cytolytic peptides called phenol-soluble modulins that have the capacity to recruit, activate and subsequently damage human neutrophils . Phenol-soluble modulin-alpha is rarely detected in HA-MRSA strains. Recently, the arginine catabolic mobile element was found in association with the staphylococcal chromosomal cassette mec in USA300 lineages, but it was absent in most of the other CA-MRSA isolates, including ST80 . This element encodes an arginine deiminase pathway and a putative oligopeptide permease operon (Opp-3). Arginine deiminase could enhance arginine catabolism and the survival of USA300 in anaerobic environments. Opp-3 may contribute to the overall fitness of USA300 by improving growth, adhesion to host cells, and expression of virulence determinants. The fitness of CA-MRSA may also be influenced by the polymorphism of the S. aureus PVL .
The possibility should be considered that the USA300 clone that has recently arrived in Europe may replace ST80, leading to a situation similar to that seen in the USA.