Methicillin-Resistant Staphylococcus aureus ST80 Induce Lower Cytokine Production by Monocytes as Compared to Other Sequence Types.

Methicillin-resistant Staphylococcus aureus (MRSA) remains an important cause of nosocomial and community-associated infections due to its ability to produce toxins and evade host's immune responses. The aim of the present study was to investigate the association of monocytes immune response in terms of cytokines produced after inoculation with different MRSA clones. Thirty-one clinical MRSA strains were selected on the basis of clonal types, accessory gene regulator (agr) groups and toxin genes carriage. Isolates were identified as S. aureus by Gram stain, catalase, coagulase production and PCR for nuc gene. The presence of mecA, lukS/lukF-PV (Panton-Valentine Leukocidin) and tst (Toxic Shock Syndrome Toxin-1) genes, as well as, the determination of agr groups was performed by PCR. Clonality was investigated by means of multi-locus sequence typing (MLST). Peripheral blood mononuclear cells were stimulated with live bacterial cells for 45 min at a ratio of 1:10. Cells were incubated for 10 h and supernatants were collected. The levels of Tumor Necrosis Factor alpha (TNFa), IL-1b, IL-8, IL-6, IL-12p40, IL-10, interferon-gamma (IFN-γ) and IL-2, were measured by Human Cytokine Multiplex Immunoassay kit. Thirteen strains were tst and 12 lukS/lukF-PV-positive. Seven strains belonged to ST80 and ST225, five to ST30 and ST239, while the remaining seven isolates were grouped together as "other." Strains belonging to ST80 induced statistically lower levels of TNFa, IL-1b, IL-8, IL-6, IL-10, IFN-γ, and IL-2. PVL-positive strains classified into ST80 clone induced statistically lower concentrations of most cytokines as compared to PVL-positive strains belonging to other clones, tst-positive strains and toxin-negative ones. Strains of agr3 group belonging to ST80 induced statistically lower concentrations of most tested cytokines as compared to agr3 strains not-belonging to ST80, agr2 or agr1. This low induction of immune response by MRSA ST80 cannot be attributed to the presence of neither lukS/lukF-PV nor agr3.

Spread of Tst-Positive Staphylococcus aureus Strains Belonging to ST30 Clone among Patients and Healthcare Workers in Two Intensive Care Units.

Staphylococcus aureus is a major cause of infections. Toxic shock syndrome toxin (TSST-1) and Panton-Valentine leukocidin (PVL) are associated with severe clinical syndromes. S. aureus colonizing isolates recovered from healthcare workers and patients in the intensive care unit (ICU) of a university hospital comprising Group A were compared with those from adult non-ICU carriers (Group B). mecA, lukS/lukF-PV (Panton-Valentine leukocidin, PVL), and tst (toxic shock syndrome toxin) gene carriage was detected by PCR. Clones were identified in all methicillin-resistant S. aureus (MRSA) and toxin-positive methicillin-susceptible strains (MSSA) by pulsed-field gel electrophoresis (PFGE), agr groups, and multi locus sequencing typing (MLST). Group A included 90 S. aureus isolates, whereas Group B 53. PVL was more frequently found among MRSA vs. MSSA (p < 0.001) and in strains of Group B as compared to Group A (p < 0.001), consistent with the spread of ST80-IV. Higher incidence of tst gene carriage was identified among MSSA vs. MRSA (P 0.005) belonging mainly to ST30, and Group A vs. Group B (P 0.002). The wide dissemination of ST80-IV mainly in the community is responsible for a high percentage of PVL-positive MRSA, while silent spread of tst-positive S. aureus clones among ICU patients and personnel poses a threat of hospital transmission and possible severe infections.

Interspecies spread of Staphylococcus aureus clones among companion animals and human close contacts in a veterinary teaching hospital. A cross-sectional study in Greece.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) prevalence among companion animals and veterinary personnel (VP) was investigated. Strains' molecular characteristics were evaluated in order to assess S. aureus transmission. Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and Etest. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (Panton-Valentine leukocidin, PVL) genes were investigated by PCR. Genotypes were identified by Multi Locus Sequence Typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), accessory gene regulator group (agr), spa and Pulsed Field Gel Electrophoresis (PFGE). S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or Coagulase-negative staphylococci co-carriage showed significantly higher prevalence of S. aureus isolation (p<0.05). Twenty-six pets and five VP carried PVL-positive S. aureus. In total, 60 S. aureus strains were recovered (53 from pets, seven from VP) of which 16 were MRSA (26.7%), 12 mecA- and four mecC-positive. MRSA showed higher resistance rates against other antimicrobials as compared to methicillin-susceptible ones. Strains were classified by MLST in 13 STs, with the predominance of ST80 and ST15. In MRSA, SCCmec types II, IV and XI were identified. The most frequent spa types were t5559 and t7558. Fifty-six strains were classified into 15 PFGE types. Comparison of genetic markers shows that identical or very similar strains disseminate among animals and VP. Companion animals harbor PVL-positive clones constituting a possible source for transmission to humans.

Activity of vancomycin, linezolid, and daptomycin against staphylococci and enterococci isolated in 5 Greek hospitals during a 5-year period (2008-2012).

The tendency of vancomycin, linezolid, and daptomycin MICs was investigated among 6920 staphylococci and enterococci during a 5-year period. Antimicrobial consumption was determined. Decrease of vancomycin MIC was detected associated with reduction in consumption. Linezolid and daptomycin remained active. An upward trend of linezolid MIC for methicillin-resistant staphylococci was observed.

Biofilm synthesis and presence of virulence factors among enterococci isolated from patients and water samples.

The goal of this study was to compare biofilm synthesis among enterococci recovered from clinical samples (infection or colonization) of patients as well as environmental samples in order to determine possible virulence factors and clonal relationship. During a two-year period, clinical samples (blood, catheter tips, bronchial secretions, wounds, peritoneal fluid, urine) and rectal swabs collected from hospitalized patients as well as environmental water samples were tested for the presence of Enterococcus faecalis and Enterococcus faecium. Antibiotic susceptibility testing was performed by the disc diffusion method and Etest. Strains were tested for the presence of vanA, vanB, esp, ace and asp genes by PCR. Clones were identified by PFGE (SmaI). From infected patients, 48 strains were identified: 24 Enterococcus faecium (10 vanA-positive, 14 vancomycin-susceptible) and 24 Enterococcus faecalis (one vanA-positive, 23 vancomycin-susceptible). Among 143 colonizing isolates, 134 were Enterococcus faecium (58 vanA-positive, 11 vanB-positive, 65 vancomycin-susceptible) and nine Enterococcus faecalis (three vanA-positive, two vanB-positive, four vancomycin-susceptible). Among 167 environmental water samples, 51 Enterococcus faecalis and 19 Enterococcus faecium isolates, all glycopeptide-susceptible, were recovered. In total, 64 strains produced biofilm, whereas 34 were esp-positive, 64 asp-positive and 54 ace-positive. Biofilm production was associated with the presence of esp (P < 0.001) and ace genes (P = 0.021), being higher in infecting (P < 0.001) and water (P 0.005) isolates as compared with colonizing ones. Clones of environmental water-strains were different than the patients' clones. The differences found in the incidence of antibiotic resistance, virulence factors and clones suggest that hospital and water enterococci are of different origin.

Coagulase-negative staphylococcal bloodstream and prosthetic-device-associated infections: the role of biofilm formation and distribution of adhesin and toxin genes.

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 (P=0.003), 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 (P=0.006 and P=0.045, 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 study suggest that the pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capabilities of S. epidermidis and S. haemolyticus strains.

Spread of multidrug-resistant Pseudomonas aeruginosa clones in a university hospital.

An outbreak of multidrug-resistant Pseudomonas aeruginosa (MDRPA) infections in a university hospital is described. Phenotypic and genotypic analysis of 240 isolates revealed that 152 patients, mainly in the intensive care unit (ICU), were colonized or infected with MDRPA, the majority with O11. All metallo-ß-lactamase (MBL)-positive isolates carried the bla(VIM-2) or bla(VIM-1) gene. One or more type III secretion system toxin genes were detected in most isolates. Five dominant pulsed-field gel electrophoresis (PFGE) types were characterized, associated with ST235, ST111, ST253, ST309, and ST639.

Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests.

Staphylococcus aureus strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which S. aureus had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to S. aureus culture supernatants. The corresponding S. aureus isolates were characterized by PCR for the presence of the PVL locus (lukS-PV and lukF-PV) and the mecA gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185 S. aureus isolates were PVL locus positive (PVL(+)); 28 of these were also mecA positive. PVL was detected in the supernatants of all PVL(+) strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL(+) strains ranged from 0 to 399 microg/ml by ELISA. By the use of 0.015 microg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by S. aureus during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.