Antimicrobial and anti-biofilm properties of new taxodione derivative from hairy roots of Salvia austriaca.

The aim of the present report was to evaluate antimicrobial/anti-biofilm activity of 7-(2-oxohexyl)-taxodione, a novel taxodione derivative isolated from n-hexane extract of Salvia austriaca hairy roots. Antimicrobial assays showed that 7-(2-oxohexyl)-taxodione was at least 4 times more active than taxodione against methicillin-susceptible as well against methicillin-resistant staphylococci with MIC of 1.25-2.5 µgml(-1). This compound was less active against vancomycin-resistant enterococci (VRE), on the same level as taxodione (MIC ranged 10.0-20.0 µgml(-1)). The presence of 7-(2-oxohexyl)-taxodione in the culture medium (at MIC, ½ MIC or » MIC) decreased adhesion of staphylococci to abiotic surfaces, which in turn caused a reduction in biofilm formation during 24h, by approximately 25-30%. Also, the extent of established biofilm eradication was found to be significant, although it required an increased concentration of the compound. This is the first report on the antimicrobial activity of this, up to now not known compound, isolated from transformed roots of S. austriaca.

Synthesis, structural characterization, antimicrobial and cytotoxic effects of aziridine, 2-aminoethylaziridine and azirine complexes of copper(II) and palladium(II).

The synthesis, spectroscopic and X-ray structural characterization of copper(II) and palladium(II) complexes with aziridine ligands as 2-dimethylaziridine HNCH(2)CMe(2) (a), the bidentate N-(2-aminoethyl)aziridines C(2)H(4)NC(2)H(4)NH(2) (b) or CH(2)CMe(2)NCH(2)CMe(2)NH(2) (c) as well as the unsaturated azirine NCH(2)CPh (d) are reported. Cleavage of the cyclometallated Pd(II) dimer [µ-Cl(C(6)H(4)CHMeNMe(2)-C,N)Pd](2) with ligand a yielded compound [Cl(NHCH(2)CMe(2))(C(6)H(4)CHMe(2)NMe(2)-C,N)Pd] (1a). The reaction of the aziridine complex trans-[Cl(2)Pd(HNC(2)H(4))(2)] with an excess of aziridine in the presence of AgOTf gave the ionic chelate complex trans-[(C(2)H(4)NC(2)H(4)NH(2)-N,N')(2)Pd](OTf)(2) (2b) which contains the new ligand b formed by an unexpected insertion and ring opening reaction of two aziridines ("aziridine dimerization"). CuCl(2) reacted in pure HNC(2)H(4) or HNCH(2)CMe(2) (b) again by "dimerization" to give the tris-chelated ionic complex [Cu(C(2)H(4)NC(2)H(4)NH(2)-N,N')(3)]Cl(2) (3b) or the bis-chelated complex [CuCl(C(2)H(2)Me(2)NC(2)H(2)Me(2)NH(2)-N,N')(2)]Cl (4c). By addition of 2H-3-phenylazirine (d) to PdCl(2), trans-[Cl(2)Pd(NCH(2)CPh)(2)] (5d) was formed. All new compounds were characterized by NMR, IR and mass spectra and also by X-ray structure analyses (except 3b). Additionally the cytotoxic effects of these complexes were examined on HL-60 and NALM-6 human leukemia cells and melanoma WM-115 cells. The antimicrobial activity was also determined. The growth of Gram-positive bacterial strains (S. aureus, S. epidermidis, E. faecalis) was inhibited by almost all tested complexes at the concentrations of 37.5-300.0 µg mL(-1). However, MIC values of complexes obtained for Gram-negative E. coli and P. aeruginosa, as well as for C. albicans yeast, mostly exceeded 300 µg mL(-1). The highest antibacterial activity was achieved by complexes 1a and 2b. Complex 2b also inhibited the growth of Gram-negative bacteria.

Bacteria competing with the adhesion and biofilm formation by Staphylococcus aureus.

The activity of antagonistic substances produced by Pseudomonas aeruginosa and Lactobacillus acidophilus against the planktonic and sessile populations of Staphylococcus aureus strains was demonstrated. The strongest effects were caused by probiotic L. acidophilus strain - bacteriocin-like inhibitory substances (BLIS) positive. However, the S. aureus A3 growth, adhesion and biofilm formation was also limited by cell-free supernatant of L. acidophilus H-1 (BLIS negative). Moreover, competitive direct interactions were observed between staphylococci and the above bacteria, which influenced the formation of dualspecies aggregates on the surface.

Subtyping of Listeria monocytogenes isolates by actA gene sequencing, PCR-fingerprinting, and cell-invasion assay.

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.

The influence of Lactobacillus acidophilus-derived surfactants on staphylococcal adhesion and biofilm formation.

The ability of surfactants obtained from three Lactobacillus acidophilus strains to inhibit Staphylococcus aureus and S. epidermidis biofilms was evaluated. Their influence was determined on bacterial initial adhesion, biofilm formation and dispersal using MTT-reduction assay, confocal laser scanning microscopy and image PHLIP analysis. The number of adhering S. aureus and S. epidermidis cells after a 3-h co-incubation with biosurfactants was reduced by 5-56 % in a strain-and dose-dependent manner. S. epidermidis-and, to a lower extent, in S. aureus-biofilm formation was also inhibited in the presence of the tested surfactants. The addition of surfactants to preformed mature biofilms accelerated their dispersal, and changed the parameters of biofilm morphology. The L. acidophilus-derived surfactants inhibit bacterial deposition rate and biofilm development (and also its maturation) without affecting cell growth probably due to the influence on the cell-surface hydrophobicity of staphylococci.

Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: salvipisone as a potential anti-biofilm agent active against antibiotic resistant Staphylococci.

The antimicrobial activities of crude dichloromethane fractions from acetone extracts of Agrobacterium rhizogenes transformed roots and roots of field-grown plants of Salvia sclarea as well as four pure abietane diterpenoids isolated from the hairy root cultures were determined. The growth of Gram-positive bacteria (Staphylococcus aureus, S. epidermidis, Enterococcus faecalis) but not Gram-negative ones (Escherichia coli, Pseudomonas aeruginosa) or pathogenic fungi (Candida albicans) was inhibited by fractions tested at concentrations of 37.5-75.0 microgml(-1). Abietane diterpenoids: salvipisone, aethiopinone, 1-oxoaethiopinone and ferruginol were shown to be bacteriostatic as well as bacteriocidal for the cultures of S. aureus and S. epidermidis strains, regardless of their antibiotic susceptibility profile. This was demonstrated by using simultaneously the optical density measuring method and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction assay. The highest activity was shown by salvipisone which demonstrated also a very interesting activity when its effect on 24-h-old staphylococcal biofilm cells viability was examined. It limited the survival of biofilms formed by S. aureus as well as by S. epidermidis, putting this compound to the list of potential anti-biofilm agents, better than most of known antibiotics.

Staphylococcus aureus biofilm as a target for single or repeated doses of oxacillin, vancomycin, linezolid and/or lysostaphin.

Due to high resistance, standard chemotherapy of biofilm-associated staphylococcal infections is ineffective and a number of alternative approaches to antimicrobial treatment have been proposed. Minimum inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) of oxacillin (Oxa), vancomycin (Van), linezolid (Lzd) and lysostaphin (Lss) as well as the possible synergistic effect of the antibiotics and lysostaphin were determined. The Lss susceptibility of Staphylococcus aureus planktonic and bio-film cultures varied and was strain-dependent. The synergistic effect of sub-BIC(Lss)+Oxa was observed for methicillin-sensitive S. aureus (MSSa) and methicillin-resistant S. aureus (MrSa), but not for heterogeneously vancomycin-resistant S. aureus (V(h)Sa) biofilm. Van with sub-BICL(Lss) was effective against M(s)Sa and MrSa biofilm, when applied in three subsequent doses. Only sub-BICL(Lss)+Lzd combination, given as three cycles therapy, was effective in disruption of all 3 (M(s)Sa, M(r)Sa, V(h)Sa) biofilms.

Superantigen types in Staphylococcus aureus isolated from patients with cystic fibrosis.

The screening of 17 SAg genes of S. aureus isolated from the sputum of cystic fibrosis (CF) patients revealed that among 47 genetically different strains, 39 (83 %) carried SAg genes. Superantigens forming enterotoxin gene cluster were detected in 20 strains. The 2nd most common superantigen type was selk detected in 13 strains. In 9 strains, selk occurred together with the sea gene. Out of 74 strains recovered from nasal carriers, 56 (75 %) were found to carry SAg genes, 38 carried egc genes, while selk was detected in 5 strains. The predominant SAg types in both investigated S. aureus populations were egc and selk/sea, but selk gene frequency was significantly higher in the CF-derived strains.

A potential double role of anti-Lewis X antibodies in Helicobacter pylori-associated gastroduodenal diseases.

In this study, we found Lewis X (Le(x)) determinants on 68% of Helicobacter pylori isolates from patients with chronic gastroduodenal diseases. Anti-Le(x) IgG were detected more frequently in the sera from dyspeptic children and adults (45 and 46%), with or without proved (culture) H. pylori infection, than in the sera from healthy individuals (14% and 25%). In contrast, the prevalence of anti-Le(x) IgM was higher in the groups of healthy individuals than in the groups of dyspeptic patients. Moreover, anti-Le(x) monoclonal antibody of IgM class enhanced the uptake of Le(x)(+) but not Le(x)(-) H. pylori isolates by phagocytes. In the sera from some dyspeptic patients, we detected Le(x)-anti-Le(x) IgG immune complexes (Le(x) ICs). There was a great difference between children and adults as regards the presence of Le(x) ICs. The immune complexes were found in the sera from nine out of 29 (27%) H. pylori-infected and three out of eight (37%) uninfected adult dyspeptic patients. In comparison, Le(x)-anti-Le(x) IgG ICs were detected only for two out of 18 (11%) H. pylori-infected children. Le(x) ICs were not found in the sera from healthy individuals. Our results suggest that anti-Le(x) IgM may play a protective role in H. pylori infections. In contrast, anti-Le(x) IgG and particularly Le(x)-anti-Le(x) IgG ICs might contribute to the pathogenesis of chronic H. pylori infections.

Characterization of Staphylococcus aureus strains isolated from cystic fibrosis patients by conventional and molecular typing.

The phenotypic and genotypic characteristics of Staphylococcus aureus strains isolated from respiratory tract of cystic fibrosis (CF) patients were investigated. Slime production, cell-surface hydrophobicity, type of capsular polysaccharide, profile of heteroresistance to methicillin and Sma I restriction profiles were evaluated. S. aureus CF strains have been shown to be heterogeneous in respect to several important features. All of them were slime producing with variation in colony morphology. High or moderate cell-surface hydrophobicity (CSH) was found for, respectively, 16.2% and 83.8% strains. Thirty strains were resistant to methicillin, 60% of them showed heteroresitance and 40% were homoresistant. It was found that 59.6% of strains produced capsular polysaccharides (CP) of 5 or 8 type. Among CP5/CP8 strains, CP8 was the predominant type (81.1%). Typing of 62 CF strains by macrorestriction analysis of chromosomal DNA revealed several major types, differing in their SmaI profiles with a similarity coefficient lower than 0.4. Some of the strains isolated from the same patient at different times of hospitalization, as well as strains isolated at the same time from the relatives, were identical in their PFGE pattern.

The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity.

In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF-alpha) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high-virulence hemolytic isolates induced significantly stronger TNF-alpha production by infected macrophages than the low-virulence unhemolytic bacilli.

Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods.

The aim of this work was to compare the possibility of identifying Listeria monocytogenes strains isolated from meat and sausage on the basis of the API-Listeria test, production of phosphatidylinositol-specific phospholipase C (PI-PLC) and polymerase chain reaction (PCR) for a DNA fragment of the hlyA gene encoding listeriolysin O. Forty-six strains were isolated and examined. The lethality of some Listeria isolates for BALB/c mice was also determined. In this study, all isolates identified as L. monocytogenes in the API test gave a positive signal in the PCR. Listeriae identified as L. innocua or L. welshimeri in the API test were negative in the PCR conducted with the primers for listeriolysin O. All strains identified as L. monocytogenes on the basis of the API test and the PCR produced PI-PLC. However, this activity was not limited to the bacteria of this species. Four out of 17 L. innocua and three out of 10 L. welshimeri isolates were PI-PLC-positive. None of the L. innocua or L. welshimeri isolates (neither PI-PLC+ or PI-PLC-) showed lethality for BALB/c mice. In contrast, two L. monocytogenes isolates as well as a reference L. monocytogenes strain killed all mice used for the experiment.

Saccharomyces cerevisiae IRR1 protein is indirectly involved in colony formation.

The ability of a microorganism to adhere to a solid support and to initiate a colony is often the first stage of microbial infections. To date, studies on S. cerevisiae cell-cell and cell-solid support interactions concerned only cell agglutination during mating and flocculation. Colony formation has not been studied before probably because this species is not pathogenic. However, S. cerevisiae can be a convenient model to study this process, thanks to well-developed genetics and the full knowledge of its nucleotide sequence. A preliminary characterization of the recently cloned essential IRR1 gene indicated that it may participate in cell-cell/substrate interactions. Here we show that lowering the level of expression of IRR1 (after fusion with a regulatory catalase A gene promoter) affects colony formation and disturbs zygote formation and spore germination. All these processes involve cell-cell or cell-solid support contacts. The IRR1 protein is localized in the cytosol as verified by immunofluorescence microscopy, and confirmed by cell fractionation and Western blotting. This indicates that Irr1p is not directly involved in the cell-solid support adhesion, but may be an element of a communication pathway between the cell and its surroundings.

[Detection of bacterial biofilm on medical biomaterials]. / Wykrywanie biofilmu bakteryjnego na biomaterialach medycznych.

This study was performed to assess the value TTC assay in the diagnosis of biomaterial-associated infections. In this assay, soluble colourless TTC is reduced to insoluble red formazan by electron transfer associated with active oxidative bacterial metabolism and is precipitated intracellularly. Microbial adhesion and biofilm formation on the surface of medical prosthetic devices (vesicular and urinary catheters) made of various polymers (PTFE, H-PE, PCW, SL), were determined. The microorganisms which are most often isolated in medical device-associated infections: S. aureus, S. epidermidis, E. faecalis, E. coli, P. vulgaris, P. aeruginosa, C. albicans, were included into the study. The obtained results indicate that the assay using TTC as a metabolic indicator of bacterial biofilm presence, is technically simple to conduct with minimal setup time. Even when classical cultures yielded no bacterial growth, TTC assessments demonstrated bacterial biofilms. TTC assay could be recommended as a quick routine method for confirmation of biomaterial device-associated infection.

The virulence of Staphylococcus aureus isolates differing by siderophore production.

In previous study we demonstrated that Staphylococci aureus clinical and environmental isolates differ by siderophore production (Lisiecki et al., 1997), the aim of the present study was to check a possible role of siderophore-dependent iron acquisition system of outcome of staphylococcal diseases. The systemic and local staphylococcal infections were induced in mice by inoculation of three S. aureus strains differing by siderophore production. We found that S. aureus B 47 strain characterized by enhanced siderophore activity was more virulent in both systemic and local infection models and it was more resistant to anti-bacterial activity of neutrophils than S. aureus B 63 and B 32 strains expressing weaker siderophore production. The results suggest that effective siderophore-dependent iron acquisition system may be beneficial to S. aureus strains in their pathogenic activity in vivo.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-coated implants and their potential for reducing biomaterial-associated infection in neutropenic hosts.

The incidence of infections associated with the use of medical biomaterials is high for skin-penetrating devices, when microbes of the normal skin flora like coagulase-positive and coagulase-negative staphylococci dominate as causative organisms. The most serious ones are infections in immunocompromised individuals. A mouse model of subcutaneous staphylococcal infection yielding abscesses in cyclophosphamide-induced neutropenic mice implanted with heparinized polyethylene (H-PE) was used. The present study addresses the question of the effects of implant modification with recombinant granulocyte-macrophage stimulating factor (rGM-CSF) on the course of infection. Our findings demonstrate that such modification reduces the proliferation of bacteria within the abscess and as a consequence limits the dissemination of bacteria from the local infection induced in the neutropenic host.

Specific immune response to staphylococcal antigens during long-lasting biomaterial implantation.

Biomaterial-associated infections caused by staphylococci are one of the main therapeutic problems in modern medicine. There is no doubt that local disfunction of polymorphonuclear leukocytes and macrophages predisposes to such infections. However, it is not clear how implantation of a foreign body influences the antibacterial immune response. We analyzed some parameters of the specific immune response to staphylococcal antigens, in mice implanted for 3 months with heparinized polyethylene. Three weeks before the evaluation of the immune response, mice (implanted and non-implanted) were infected i.p. with 2 x 10(7) cells of Staphylococcus aureus Cowan 1. The proliferation of splenocytes was determined on the basis of [3H]thymidine incorporation in cultures stimulated with staphylococcal lipoteichoic acid, protein A, alpha-toxin, or phytohemagglutinin. Moreover, the level of specific antibodies to staphylococcal antigens was determined in serum samples (ELISA with the antigens lipoteichoic acid, protein A, and alpha-toxin). The data obtained indicate that long-lasting implantation caused evident changes in proliferative activity of lymphocytes and in humoral response to staphylococcal antigens. It enhanced spontaneous and lipoteichoic acid- or alpha-toxin-stimulated proliferation of splenocytes, in vitro. In contrast, heparinized polyethylene-implanted animals showed a significant decrease in the production of anti-protein A IgG2b and anti-alpha-toxin IgG2a and IgG2b.

Altered immune response to staphylococcal antigens in long-lasting implanted mice.

Staphylococcal infections constitute one of the main problems associated with clinical applications of various prosthetic medical devices (biomaterials). As the magnitude of the infection risk depends often on the duration of device installation, and the incidence of infections is higher in skin-penetrating devices, we studied some parameters of specific immune response to staphylococcal antigens in mice subcutaneously (s.c.) implanted for three months with heparinized polyethylene (H-PE). Three weeks before the evaluation of immune response, mice (implanted and non-implanted) were s.c. infected with 10(7) of Staphylococcus aureus Cowan 1. The proliferation of lymph node cells was determined on the basis of 3H-thymidine incorporation in 3-days cultures stimulated with: staphylococcal lipoteichoic acid (LTA), protein A (SpA), alpha-toxin, or with phytohemagglutinin (PHA). Moreover, the levels of specific antibodies to staphylococcal antigens were determined in serum samples (ELISA against: LTA, SpA, alpha-toxin). The data obtained indicate that long-lasting implantation caused evident changes in proliferative activity of lymphocytes and humoral response to staphylococcal antigens. It enhances alpha-toxin and LTA stimulated proliferation of lymph node lymphocytes in vitro. In contrast, H-PE-implanted animals demonstrated a significant decrease in the production of anti-SpA IgG2a and IgG2b and increase in the synthesis of anti-LTA IgG1 antibodies.

The host response to Listeria monocytogenes mutants defective in genes encoding phospholipases C (plcA, plcB) and actin assembly (actA).

Several genes involved in the determination of Listeria monocytogenes pathogenesis have been identified. Among them, plcA gene encodes phosphatidylinositol-specific phospholipase C (PI-PLC), plcB gene encodes a broad-range phospholipase C (PC-PLC), and actA encodes a protein contributing to actin assembly in infected cells. The interaction of L. monocytogenes wild type (LO 28) strain and two derivative mutants, plcA- (BUG 206) and actA-/plcB- (LUT 12), with macrophages and T lymphocytes was investigated in a mouse model of listeriosis. Both mutants showed evidence of attenuation. The plcA- mutant, but not the plcB- mutant, expressed an increase in susceptibility to the anti-listerial activity of macrophages. Both mutants showed a decreased ability to induce IL-12 production by bone marrow macrophages when co-stimulated with E. coli LPS or IFN-gamma. In vivo, L. monocytogenes plcA- mutant was found to be a more effective stimulator of T cells than the wild LO 28 strain.

The immune response to staphylococcal antigens in mice depleted of macrophages by Cl2MDP-liposomes.

To investigate the role of macrophages in the induction of the production of antibody to staphylococcal antigens, we used Cl2MDP (clodronate) liposomes as a tool for local macrophage depletion. Macrophage depletion caused in mice by intraperitoneal (i.p.) injection of Cl2MDP liposomes was associated with a reduction in the clearance of Staphylococcus aureus Cowan 1 bacteria from the tissues of infected animals and with a marked decrease in the bactericidal activity of macrophages escaping from the lethal effect of clodronate. Despite the functional defect of macrophages, the mice treated with Cl2MDP liposomes two days before the injection of alpha-toxin (toxoid) or whole heat-killed S. aureus Cowan 1 bacteria, demonstrated an enhancement in the production of anti-staphylococcal alpha-toxin IgM and anti-collagen-binding protein IgG. A similar enhancement of antistaphylococcal antibody synthesis was observed in mice after receiving phosphate buffered saline (PBS) encapsulated in liposomes.