Fevronia Kolonitsiou

An extracellular Staphylococcus epidermidis polysaccharide: relation to Polysaccharide Intercellular Adhesin and its implication in phagocytosis

BackgroundThe skin commensal and opportunistic pathogen Staphylococcus epidermidis is a leading cause of hospital-acquired and biomaterial-associated infections. The polysaccharide intercellular adhesin (PIA), a homoglycan composed of β-1,6-linked N-acetylglucosamine residues, synthesized by enzymes encoded in icaADBC is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated S. epidermidis infection. Extracellular mucous layer extracts of S. epidermidis contain another major polysaccharide, referred to as 20-kDa polysaccharide (20-kDaPS), composed mainly out of glucose, N-acetylglucosamine, and being partially sulfated. 20-kDaPS antiserum prevents adhesion of S. epidermidis on endothelial cells and development of experimental keratitis in rabbits. Here we provide experimental evidence that 20-kDaPS and PIA represent distinct molecules and that 20-kDaPS is implicated in endocytosis of S. epidermidis bacterial cells by human monocyte-derived macrophages.ResultsAnalysis of 75 clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kDaPS is expressed exclusively in S. epidermidis but not in other coagulase-negative staphylococcal species. Tn917-insertion in various locations in icaADBC in mutants M10, M22, M23, and M24 of S. epidermidis 1457 are abolished for PIA synthesis, while 20-kDaPS expression appears unaltered as compared to wild-type strains using specific anti-PIA and anti-20-kDaPS antisera. While periodate oxidation and dispersin B treatments abolish immuno-reactivity and intercellular adhesive properties of PIA, no abrogative activity is exerted towards 20-kDaPS immunochemical reactivity following these treatments. PIA polysaccharide I-containing fractions eluting from Q-Sepharose were devoid of detectable 20-kDaPS using specific ELISA. Preincubation of non-20-kDaPS-producing clinical strain with increasing amounts of 20-kDaPS inhibits endocytosis by human macrophages, whereas, preincubation of 20-kDaPS-producing strain ATCC35983 with 20-kDaPS antiserum enhances bacterial endocytosis by human macrophages.ConclusionsIn conclusion, icaADBC is not involved in 20-kDaPS synthesis, while the chemical and chromatographic properties of PIA and 20-kDaPS are distinct. 20-kDaPS exhibits anti-phagocytic properties, whereas, 20-kDaPS antiserum may have a beneficial effect on combating infection by 20-kDaPS-producing S. epidermidis.

A newly described vancomycin-resistant ST412 Enterococcus faecium predominant in Greek hospitals

A total of 359 vancomycin-resistant enterococci (344 Enterococcus faecium and 15 E. faecalis) collected during 2007 from eight tertiary-care hospitals in Greece were analysed for genotypic characteristics. Four common clones, ST412, ST203, ST16 and ST17, were identified among E. faecium and one clone, ST28, among E. faecalis strains.

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.

In vitro activity of daptomycin against Gram-positive cocci: the first multicentre study in Greece

A total of 10420 Gram-positive cocci (including staphylococci, enterococci and various groups of streptococci) collected from clinically significant specimens in ten Greek hospitals during 2006--2007 were tested for their susceptibility to daptomycin. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. Daptomycin demonstrated very high activity against Enterococcus faecalis (MIC at which 50% of the isolates were inhibited (MIC50) = 1mg/L and MIC at which 90% of the isolates were inhibited (MIC90) = 1.36 mg/L), Enterococcus faecium (MIC50 = 1.36 mg/L and MIC90 = 1.90 mg/L), Streptococcus pyogenes (MIC50 = 0.12 mg/L and MIC90 = 0.50mg/L), Streptococcus agalactiae (MIC50 = 0.09 mg/L and MIC90 = 0.12 mg/L), Streptococcus pneumoniae (MIC50 = 0.24 mg/L and MIC90 = 0.5 mg/L) and viridans group streptococci (MIC50 = 0.50 mg/L and MIC90 = 0.89 mg/L). Resistance to linezolid and vancomycin for enterococci and to penicillin for streptococci appears to be independent of reduced susceptibility to daptomycin. On the other hand, daptomycin was also active against meticillin-resistant Staphylococcus aureus (MIC50 = 0.44 mg/L and MIC90 = 0.78 mg/L) and meticillin-resistant coagulase-negative staphylococci (MIC50 = 0.24 mg/L and MIC90 = 0.44 mg/L); however, 0.9% of the staphylococci tested had an MIC > 1mg/L, which is the Clinical and Laboratory Standards Institute breakpoint proposed for susceptibility. For all tested organism groups, resistance to daptomycin was not associated with glycopeptide resistance.

Immunoreactivity of 80-kDa peptidoglycan and teichoic acid-like substance of slime producing S. epidermidis and specificity of their antibodies studied by an enzyme immunoassay

S. epidermidis is considered an important cause of nosocomial bacteraernia in immunocompromized hosts as well as the commonest agent of sepsis in patients with prosthetic devices. Pathogenesis is attributed to adherence and growth on bioniaterials facilitated by production of extracellular slime. The major macromolecules of slime are: a 20-kDa acidic polysaccharide (20-kDa PS) comprising the 60% of carbohydrate-containing slime macromolecules, a peptidoglycan with average molecular size of 80-kDa (30% of slime dry weight) and cell wall teichoic acid-like substance. In this study, antibodies to these macromolecules as well as crude slime were raised in rabbits and their immunological reactivity and specificity were studied by an enzyme immunoassay. All isolated macromolecules induced the production of specific antibodies. 20-kDa PS was less immunogenic than 80-kDa peptidoglycan and teichoic acid-like substance. However, 20-kDa PS was the most potent inhibitor of the reaction of slime with its homologous antibodies revealing that this polysaccharide is the major antigenic determinant of slime. All three antibodies specifically recognize (p < 0.05) and react with slime-producing S. epidermidis in comparison to other staphylococci species. Obtained results indicate that the 20-kDa PS may be distributed in the surface of the slime exposing most of its antigenic determinants to the immune system, whereas those of 80-kDa peptidoglycan and teichoic acid-like substance seem to be less accessible.

Linezolid-Resistant Enterococci in Greece: Epidemiological Characteristics

Background: 14 linezolid-resistant enterococci (6 Enterococcus faecium and 8 Enterococcus faecalis) collected during 2009 from patients hospitalized in intensive care units of different Greek hospitals were investigated. Methods: The mechanism of resistance to linezolid was determined by sequencing analysis of the domain V of 23S rDNA, while the clonal relatedness was defined by pulsed-field gel electrophoresis and multilocus sequence typing. Results: All linezolid-resistant enterococci carried the G2576T mutation. E. faecium belonged to the international epidemic clones ST16, ST17, ST203 and ST65, while all E. faecalis strains belonged to the ST28 clone. Conclusions: The spread of common linezolid-resistant enterococcal clones in intensive care units located in different areas of Greece emphasizes the importance of application of infection control measures to prevent the spread of such strains.

In vitro activity of tigecycline and colistin against A. baumannii clinical bloodstream isolates during an 8-year period

Abstract Acinetobacter baumannii has emerged as an important and problematic pathogen causing bloodstream infections (BSI) in hospitalized patients. Results of an 8-year period from a university hospital are presented. Identification of A. baumannii was performed by Gram-negative BD BBL Crystal ID and VITEK®2 system, whereas, susceptibility testing by VITEK2, Kirby–Bauer disc system, and Etest strips. Interpretation of results was based on CLSI criteria and, regarding tigecycline, Food and Drug Administration (FDA) criteria. Between 2006 and 2013, 441 among 7088 BSI cases were attributed to A. baumannii. Of all isolates, 92·1% were resistant to more than three classes of antibiotics and 79·4% were resistant to all but one or two categories of antimicrobials. Resistance to ampicillin–sulbactam, meropenem, gentamicin, ciprofloxacin, minocycline, and tigecycline increased during the study period (P<0·05). Although tigecycline resistance was low during the first 4 years of the study (25·5%), it increased up to 66·5% during 2010–2013. No isolate was colistin resistant.

Potential use of solid phase immunoassays in the diagnosis of coagulase-negative staphylococcal infections.

Staphylococcus epidermidis is a major nosocomial pathogen, even though it is a member of the normal bacterial flora of skin and the mucous membranes. A major complication is the development of biofilms on implanted medical devices. Diagnosis of coagulase-negative staphylococcal infections relies on the presence of clinical manifestation of infections and on microbiologic evidence, usually obtained after the removal of the biomaterial. Solid-phase immunoassays have not yet been used for routine diagnosis of coagulase-negative staphylococcal infections and distinction between pathogenic and normal cocci. The enzyme immunoassays developed in the last decade are presented in this review article. Serodiagnosis has been attempted by determining antibodies against bacterial cells, mixtures of S. epidermidis slime antigens and discrete slime antigens. Detection or typing of staphylococcal cells has been performed by specific antibodies and lectins. There is still a long way until the application of such assays in the routine clinical laboratory and large clinical studies are necessary.

Levels of specific antibodies towards the major antigenic determinant of slime-producing Staphylococcus epidermidis determined by an enzyme immunoassay and their protective effect in experimental keratitis.

Staphylococcus epidermidis is an important cause of bacterial keratitis. Certain S. epidermidis strains produce an extracellular slime layer rich in an acidic polysaccharide with a molecular size of 20 kDa (20-kDa PS). We have demonstrated that the level of 20-kDa PS-specific antibodies significantly rises after establishment of slime-producing S. epidermidis bacteraemia and, furthermore, that rabbit polyclonal antibodies to 20-kDa PS opsonize cells of slime-producing S. epidermidis to a great degree and promote their clearance by polymorphonuclear cells (Arch. Biochem. Biophys. 342 (1997) 389; J. Pharm. Biomed. Anal. 22 (2000) 1029). The purpose of this study was to examine the protective and therapeutic effects both of active immunization, using 20-kDa PS as antigen, and of passive administration of specific antibodies towards the 20-kDa PS in a rabbit keratitis model. For active immunization, 20 rabbits were subcutaneously immunized with 20-kDa PS, whereas for passive immunization specific polyclonal IgG antibodies against 20-kDa PS were administered to 20 rabbits 1 day before induction of infection. Clinical observations were made weekly for 1 month and levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by an enzyme immunoassay. The levels of specific anti-20-kDa PS IgG in serum and aqueous humor following either active or passive immunization were significantly higher as compared with control groups (P<0.001). Although, actively immunized rabbits showed significantly less corneal damage than control animals, passively immunized ones were significantly better protected as compared with both control and those actively immunized. Obtained results suggest that 20-kDa PS plays crucial role in the pathogenesis of S. epidermidis keratitis and that both types of immunization significantly protect against corneal S. epidermidis pathology and damage.

Immunization with specific polysaccharide antigen reduces alterations in corneal proteoglycans during experimental slime-producing Staphylococcus epidermidis keratitis

Purpose: Staphylococcus epidermidis is a leading cause of bacterial keratitis associated with corneal damage. Corneal integrity is closely associated with matrix macromolecules, such as proteoglycans (PGs) and collagen. The aim of this study was to examine whether active immunization (AI) using a major immunogenic polysaccharide determinant of slime (20-kDa PS) as antigen, and passive immunization (PI) after administration of specific antibodies toward 20-kDa PS affect the distribution of PGs as well as corneal lesions in an experimental model of slime-producing S. epidermidis keratitis. Methods: For AI, seven rabbits were immunized with 20-kDa PS, whereas for PI, seven rabbits received specific antibodies against 20-kDa PS. Lesions were graded clinically for a 21-day period. Levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by ELISA. The distribution of certain extracellular matrix PGs during corneal healing was analyzed immunohistochemically. Results: Levels of specific anti-20-kDa PS antibodies in serum and aqueous humor obtained after either AI or PI were significantly higher as compared with those in the respective nonimmunized control groups (p < 0.001). Clinical grading showed that both AI and PI rabbits had a significantly less corneal damage as compared with infected nontreated rabbits. Immunohistochemical analyses for PGs exhibited significant differences to the wounded regions as compared with noninfected corneal tissue. Accumulation of keratan sulfate PGs and decorin was observed in the corneal stroma of infected rabbits and of heparan sulfate PGs around the new-formed vessels. This phenomenon was significantly reduced in immunized animals in accordance with macroscopically decreased corneal damage observed in these animals. Conclusions: Results of this study suggest a key role of 20-kDa PS and its antibodies as prophylactic and therapeutic agents in keratitis caused by slime-producing S. epidermidis.