Three collections of Staphylococcus aureus isolates (n = 1,058) were investigated to assess the spread of Panton-Valentine leukocidin (PVL)-producing strains in Greece and their association with skin and soft-tissue infections (SSTIs). The isolates were collected during 2001-2003 from inpatients and outpatients with invasive infections in two distinct geographical areas. Clonal types were identified according to their ClaI-mecA::ClaI-Tn554::pulsed-field gel electrophoresis patterns, and the presence of the lukS-PV and lukF-PV genes was assessed by PCR. In total, 287 (27%) S. aureus isolates carried the PVL genes: 45% of methicillin-resistant S. aureus (MRSA) and 12% of methicillin-sensitive S. aureus (MSSA). All the PVL-positive MRSA isolates belonged to a single clone that was disseminated in the community and hospitals. The PVL-positive MSSA isolates were polyclonal, with 14 of 65 isolates being associated with hospital-acquired infections. The community-acquired isolates were from SSTIs, while the hospital-acquired isolates were associated with surgical wound infections, especially those involving prosthetic devices. Thus, a unique clone of PVL-positive MRSA has spread in both the community and the hospital setting in Greece, and has replaced older clonal types.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) infections. Severe MRSA infections have been associated with the virulence factor Panton-Valentine leukocidin (PVL). The aim of this study was to investigate susceptibility patterns, the presence of toxin genes, including that encoding PVL, and clonality among MRSA isolates collected from patients in Greece over a 12-year period. MRSA isolates were collected from January 2001 to December 2012 from six different hospitals. Antibiotic susceptibility was determined with the disk diffusion method and the Etest. The presence of the toxic shock syndrome toxin-1 gene (tst), the enterotoxin gene cluster (egc) and the PVL gene was tested with PCR. The genotypic characteristics of the strains were analysed by SCCmec and agr typing, and clonality was determined with pulsed-field gel electrophoresis and multilocus sequence typing. An increasing rate of MRSA among S. aureus infections was detected up to 2008. The majority of PVL-positive MRSA isolates belonged to a single clone, sequence type (ST)80-IV, which was disseminated both in the community and in hospitals, especially during the warmest months of the year. Carriage of tst was associated with ST30-IV, whereas egc was distributed in different clones. CA-MRSA isolates were recovered mainly from skin and soft tissue infections, whereas HA-MRSA isolates were associated with surgical and wound infections. During the period 2001-2012, ST80-IV predominated in the community and infiltrated the hospital settings in Greece, successfully replacing other PVL-positive clones. The predominance of ST239-III in HA-MRSA infections was constant, whereas new clones have also emerged. Polyclonality was statistically significantly higher among CA-MRSA isolates and isolates from adult patients.

An increasing number of infections caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) carrying the Panton-Valentine leukocidin (PVL) genes was recently identified in Greece. In the present study, 170 patients with S. aureus infections and 123 uninfected children (<15 years old) who had been tested for nasal carriage were evaluated during a 2-year period. The MecA, PVL and superantigen family genes, and MRSA clones, were investigated by molecular methods. Sites of infection and laboratory findings for patients were recorded. The results were compared and statistically analysed. Among 123 uninfected children 73 (59%) carried S. aureus, including four MRSA strains. Of these, three MRSA and three methicillin-sensitive S. aureus (MSSA) strains were PVL-positive (p <0.0001). Ninety-six patients (96/170) exhibited skin and soft-tissue infections (SSTIs), and 74 exhibited invasive infections. The incidence of staphylococcal infections increased during July to September each year. In total, 110 S. aureus isolates were PVL-positive (81 from SSTIs and 29 from invasive infections, p <0.0001). Ninety-nine out of 106 MRSA (93%) isolates from 170 patients carried the PVL genes (p <0.0001); 97 belonged to the clonal complex CC80. Leukocyte and polymorphonuclear cell counts were higher among children with MRSA infections (p <0.005). MSSA predominated among patients with invasive infections (43/74), and carried mainly genes of the superantigen family. Children <5 years of age showed a higher risk of MRSA infection. The present study demonstrates that infections due to PVL-positive CA-MRSA spread easily among children, and SSTIs can lead to invasive infections. Nasal colonization may be an additional factor contributing to the emergence of CA-MRSA.

Aim:  Absolute and relative quantitative real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin‐1 (TSST‐1), among methicillin‐resistant Staphylococcus aureus (MRSA). Methods and Results:  Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real‐time RT‐PCR and the SYBR® Green I. Absolute RT‐PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2−ΔΔCt and the Pfaffl analysis methods. Conclusions:  tst gene expression was best calculated by the relative real‐time RT‐PCR analysis applying the Pfaffl analysis method, taking into account the reactions’ efficiencies. Level of tst expression was related to patients’ infection and did not depend on the MRSA genetic profile. Significance and Impact of the Study:  The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real‐time RT‐PCR is more adequate.

Analysis of 177 methicillin-resistant Staphylococcus aureus (MRSA) strains for the presence of egc and tst revealed that 60 strains carried at least one of the tested genes. MRSA strains were classified by pulsed-field gel electrophoresis into four clones belonging to agr groups I and III. Toxin genotypes were related by clonal type and agr group.The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) has posed a clinical threat worldwide. Staphylococcal enterotoxins, enterotoxin-like superantigens, and the toxic shock syndrome toxin 1 that belong to pyrogenic toxin superantigens (PTSAgs) are considered major virulence factors (5, 11). The enterotoxin gene cluster (egc) includes five enterotoxin-like superantigen genes (seo, sem, sei, sen, and seg) and two pseudogenes (ent1 and ent2) (6,8,11). Recently, a new toxin, SEU, encoded by the egc operon (egc2), was identified to result from sequence divergence in the region of the pseudogenes ent1/ent2 (9).Expression of virulent genes in S. aureus is controlled by the accessory gene regulator (agr), with a polymorphism in the sequence of the autoinducing peptide and its receptor, according to which clinical strains can be divided into four agr groups (I to IV) (7, 10). S. aureus strains causing specific syndromes were linked to certain agr types (7). Thus, the purpose of the present study was to investigate the presence of the egc operon and tst in a set of 177 MRSA isolates collected from different patients admitted to the University Hospital of Patras, Greece, during 2001 to 2003, to determine the clones of isolates, and to correlate them with the agr groups and toxin gene profile.(These results were partially presented as a poster at the 15th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 2 to 5 April 2005.)All 177 MRSA isolates were tested by the disk diffusion method against various antistaphylococcal agents (12). Oxacillin MICs were determined by the agar dilution method (13), and the presence of the mecA gene was determined by PCR (14).Clones were defined by pulsed-field gel electrophoresis of SmaI digests of chromosomal DNAs performed in a CHEFF DR III apparatus (Bio-Rad Laboratories, Richmond, California) according to published protocols (4, 15) and were compared to previously identified clones existing in our hospital (1).agr typing was carried out by PCR as described previously (10) with modified thermal conditions as follows: predenaturation step for 5 min at 94°C, 25 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, extension for 1 min at 72°C, and a final extension step for 7 min at 72°C (J. Etienne and M. Bes, personal communication). Fri913 (agr class I), Fri137 (agr class II), ATCC 49775 (agr class III), and A920211 (agr class IV) were used as reference strains.The sequences corresponding to seo, sem, sei, ent1/ent2 (ent), sen, and seg included in the egc1 operon and tst were detected by PCRs (6, 7). Fri137 and Fri913, carrying the egc2 operon and tst, respectively, wer...