Abstract: To date, most animal studies of myocardial ischemia have used open-chest models with direct surgical coronary artery ligation. We aimed to develop a novel, percutaneous, minimally-invasive, closed-chest model of experimental myocardial infarction (EMI) in the New Zealand White rabbit and compare it with the standard open-chest surgical model in order to minimize local and systemic side-effects of major surgery.
Abstract: Staphylococcus epidermidis is a leading cause of hospital-acquired and biofilm-associated infections. Interactions of peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages with planktonic or biofilm phase S. epidermidis cells were studied. Biofilm phase bacteria exhibited higher attachment, as well as, a 10-fold higher intracellular survival in monocyte-derived macrophages than their planktonic counterparts. Stimulation of PBMCs and monocyte-derived macrophages was performed with live or formalin-fixed bacterial cells. Supernatant concentration of selected cytokines was measured by Luminex(®) xMAP(™) technology at different time points. As compared to planktonic phase, biofilm phase bacteria elicited lower amounts of proinflammatory cytokines and Th1 response cytokines, such as TNFα, IL-12p40, IL-12p70 and IFN-γ, whereas they enhanced production of IL-8, GM-CSF and IL-13. This phenomenon was independent of formalin pretreatment. Taken together, these results may contribute to interpretation of observed silent course of biofilm-associated infections.
Abstract: Bacterial adherence to eukaryotic cells is highly contributing to microbial pathogenesis. Bacterial adhesins, macromolecules, and glycosaminoglycan chains of the endothelial cell surface have been implicated in staphylococcal attachment. Our research group has isolated an antigenic polysaccharidic component of Staphylococcus epidermidis extracellular layer, known as 20-kDa PS (PS), and showed that antibodies against this polysaccharide protect from infections. Therefore, the role of PS in S. epidermidis adherence to endothelial cells was studied. For this purpose we examined the impact of PS on the ability of two S. epidermidis strains (a PS-producing and a non-PS-producing strain) to adhere to human endothelial cells in the presence or absence of specific antibodies to this polysaccharide. Hence, it is established that exogenous chondroitin sulfate (CS) decreases, in part, the S. epidermidis' attachment to endothelial cells and the antagonistic binding effect of CS and PS was also studied. The results obtained demonstrate that PS facilitates the adherence of S. epidermidis to both strains. CS abolished the PS-induced adherence in PS-producing strain and partially in the non-PS-producing one. Conclusively, the adherence of S. epidermidis to human endothelial cells is associated with its extracellular PS component and it is suggested that the bacterial binding via glycosaminoglycan chains is an important mechanism underlining the PS-induced binding to endothelial cells.
Abstract: The extracellular slime of Staphylococcus epidermidis contains, amongst various macromolecules, an acidic polysaccharide (PS) of a molecular mass of 20 kDa with significant antigenic and biological properties. The isolation procedure used so far includes multiple fractionations in anion-exchange chromatographic columns before its final purification by gel filtration chromatography. This protocol is laborious, time-consuming and includes the risk of unnecessary loss of PS quantities. Because of the significance of this PS, a modified protocol resulting in an easier and quicker isolation procedure was developed. Furthermore, identification, purity, charge density and molecular integrity of the isolated polysaccharide were evaluated by a reverse-polarity capillary electrophoresis method.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Methicillin-resistant coagulase-negative staphylococci (MR-CNS) (n = 132), isolated from pre-term neonates, were analysed to determine their antibiotic resistance patterns, clonal distribution, biofilm production and the presence of the ica operon. All MR-CNS were multiresistant, and 89% produced slime. A major clone was identified (77 isolates) among 115 Staphylococcus epidermidis isolates. Ten of 16 Staphylococcus haemolyticus isolates also belonged to a single clone. Most (80%) slime-positive isolates possessed all the ica genes tested, while the remaining 23 (20%) had a variety of gene combinations. The entire ica cluster was detected in three of 15 slime-negative isolates. One major and two minor slime-positive, multiresistant MR-CNS clones had disseminated among hospitalised pre-term neonates.
Abstract: The purpose of this study was to evaluate clinical and laboratory findings, treatment modalities and final outcomes of brucellosis in children and to compare our data with those of other studies performed in Greece. Fifty-two children treated for brucellosis in the Department of Pediatrics during the decade 1995-2004 were analyzed. Of the 52 children, 47 were reexamined during July 2005. Fever, arthritis or arthralgia, hepatomegaly and splenomegaly were the main findings. Young children had positive blood cultures and lower or negative antibody titers statistically significantly more often than did older children. Brucella abortus was isolated in 9 of 18 patients with positive blood cultures. Antibiotic treatment lasted for 28 days on average. There were no complications or relapses, except one, and the final outcomes were excellent.
Abstract: The purpose of the study was to compare bacterial species, clinical, laboratory and imaging findings ((99m )Tc-dimercaptosuccinic acid renal scan and voiding cystogram) in infants and children with high (>/=10(5) colony forming units (CFU)/ml, group A patients) and low (</=5 x 10(4) CFU/ml, group C patients) bacterial count in urine cultures during first episode of urinary tract infection. Group B included patients with intermediate CFU/ml. Included were 419 symptomatic patients with: (a) no previous antibiotic treatment, (b) urine samples for quantitative cultures taken by bladder catheterisation or suprapubic bladder aspiration, (c) growth of only one microorganism, and (d) age </=54 months (age of the oldest patient of group C). Out of 419 cultures, Escherichia coli grew in 315 (75.2%), gram-negative bacteria except E. coli in 91 (21.7%) and gram-positive in 13 (3.1%). Group C patients were significantly ( P <0.0001) more often affected with gram-negative pathogens except E. coli than group A patients (21/44 versus 67/360). Most of group C patients were younger than 24 months of age; none was older than 54 months. Comparison of the prevalence of clinical and laboratory (leucocyte count, CRP, ESR) findings between groups A and C showed no significant differences. There was no statistically significant difference in the prevalence of pyelonephritis, reflux and urological malformations (except reflux) between groups A and C. CONCLUSION: Low bacterial count urinary tract infections mainly affect infants and young children and are often due to gram-negative bacteria other than E. coli. Clinical and laboratory findings, prevalence of pyelonephritis, reflux and urological malformations are similar in high and low bacterial count urinary tract infections.
Abstract: The aim of this retrospective study was to assess the incidence and aetiology of central and peripheral venous catheter (C/PVC) infections during a 2-year period (1999-2000) and to determine the susceptibility of isolated microorganisms to various antimicrobial agents. Catheter tips were processed using the semiquantitative method and blood cultures were performed with the BacT/Alert automated system. Antibiotic susceptibilities were performed by disk agar diffusion and MICs were determined by Etest, according to NCCLS standards. During the study period, samples from 1039 C/PVC infections were evaluated, yielding 384 (37.0%) positive cultures. Blood cultures were also available from 274 patients, of which 155 (56.6%) yielded the same microorganism as from the catheter. No bloodstream infections were detected in 104 C/PVC-positive cases. Methicillin-resistant coagulase-negative staphylococci were the most frequent isolates, followed by Gram-negative bacteria, especially Pseudomonas aeruginosa. Resistance to glycopeptides among staphylococci and enterococci was not detected, whereas 60% of Gram-negative bacilli were resistant to beta-lactams.
Abstract: 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.
Abstract: Enterococci and especially glycopeptide-resistant strains (GRE) are widely distributed in the hospital environment, by acquiring resistance determinants and virulence factors.
Abstract: 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.
Abstract: Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS). Analysis was performed using 20 mM borare, pH = 9.3, as operating buffer and detection of the MDA-TBAS adduct at 267 nm. The method has a linear range up to 80 microM with a detection limit of 0.2 microM. The method was applied to the analysis of MDA in plasma of healthy adults, normal-gestation infants and of preterm neonates. Plasma proteins were successfully removed following centrifugation through a centricon-3 membrane. Results showed that the method can be easily and accurate applied for the determination of MDA in human plasma and that the level of MDA in pretern neonates is significantly higher (p <or= 0.001) as compared to the two other cases. This suggests that MDA analysis may be useful to monitor the development and process of diseases related to lipid peroxidation, oxidative stress and prematurity.
Abstract: 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.
Abstract: Twenty-four serum samples from multitransfused patients with beta-thalassaemia major and fourteen positive control samples, (eight haemodialysis patients and six sporadic cases of HCV infection) were selected as anti-HCV-positives by a second-generation ELISA, a confirmatory test (Abbott), and an immunoblot assay (INNO-LIA HCV AbIII, Omicron Medical). Subsequently, by means of the nested polymerase chain reaction technique (PCR), using the set of PT1-4 primers, the RNA of the virus was detected in a total of 29 samples: 22 out of 24 patients with beta-thalassaemia and 7 out of 14 from the control group were positive for the RNA of the virus. The findings show that there is a statistically significant prevalence [chi2 = 6.344, P < or = 0.02] of HCV viraemia in the population of beta-thalassaemia major in Greece, as compared with the positive control group.
