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.

Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and Staphylococcus haemolyticus, have emerged as opportunistic pathogens in immunocompromised patients and those with indwelling medical devices. In this study, CNS recovered from patients with bloodstream infections (BSIs) or prosthetic-device-associated infections (PDAIs) were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, and carriage of adhesin and toxin genes. A total of 226 CNS isolates (168 S. epidermidis and 58 S. haemolyticus) recovered from hospital inpatients with BSIs (100 isolates) or PDAIs (126 isolates) were tested for biofilm formation, antimicrobial susceptibility, and mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, sec) genes. The selected CNS were classified into pulsotypes by PFGE and assigned to sequence types by multilocus sequence typing. In total, 106/226 isolates (46.9 %) produced biofilm, whereas 150 (66.4 %) carried the ica operon. Most isolates carried mecA and were multidrug resistant (90.7 %). CNS recovered from BSIs were significantly more likely to produce biofilm (P50.003), be resistant to antimicrobials and carry mecA (P,0.001), as compared with isolates derived from PDAIs. CNS from PDAIs were more likely to carry the aap and bap genes (P50.006 and P50.045, respectively). No significant differences in the carriage of toxin genes were identified (P.0.05). Although PFGE revealed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by multilocus sequence typing revealed three major clones (ST2, ST5 and ST16). A clonal relationship was found with respect to antimicrobial susceptibility and ica and aap gene carriage, reinforcing the premise of clonal expansion in hospital settings. The results of this 3Present address: Central Veterinary Institute of Wageningen UR, Lelystad, the Netherlands.

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.

BackgroundBiofilms are considered the key factor in the development of implant-related infections. However, only a few reports have dealt with the ability of organisms isolated from such infections to develop biofilms in vitro.MethodsWe evaluated different phenotypic techniques (2 microtiter plate assays and confocal laser scanning microscopy (CLSM) and genotypic techniques (detection of the ica operon) related to biofilm development by clinical isolates of Staphylococcus spp.ResultsAll 26 strains tested (from 23 specimens) were biofilm producers. Stepanovic test detected biofilm formation in 85% of the strains, microtiter plate assay in 65%, and CLSM in 39%. The ica operon was detected in 73% of all strains (all 13 S. aureus strains and 6 of the 13 coagulase-negative Staphylococcus strains). 7 ica-negative strains were biofilm-positive by phenotypic methods.InterpretationThe detection of ica genes could not be related to the phenotypic ability of the strains to develop a biofilm in vitro, so both studies (genetic and phenotypic) are required for a better evaluation of the biofilm-producing ability of clinical strains of Staphylococcus isolated from orthopedic infections.