In vivo detection of Staphylococcus aureus in biofilm on vascular prostheses using non-invasive biophotonic imaging.

OBJECTIVES: Biophotonic imaging was compared to standard enumeration method both for counting Staphylococcus aureus in biofilm and bacterial susceptibility tests of different graft materials. DESIGN: Prospective, randomized, controlled animal study. MATERIAL AND METHODS: Five types of vascular grafts were placed subcutaneously in 35 mice and challenged with bioluminescent S. aureus. The mice were divided into equal groups as follows: group A (polyester), group B (polytetrafluoroethylene), group C and D (two types of silver acetate-coated polyester) and group E (bovine pericardium). Controls were given only the bacteria. The bioluminescence signal of S. aureus, able to predict number of viable bacteria in biofilm without any manipulation, was measured at different time points. Five days postinfection, regular cultures of adherent bacteria on grafts were obtained. Comparative analyses between bioluminescence activity and culture enumeration were performed. RESULTS: The number of viable bacteria on silver-coated prostheses was the slightest, indicating superior bacterial resistance. The density of bacteria on polytetrafluoroethylene and polyester was comparable, with a non-significant advantage for polytetrafluoroethylene. Moreover, bioluminescence detected the number of viable S. aureus in biofilm more exactly compared to enumeration of bacteria. CONCLUSION: Bioluminescence imaging can be considered a useful tool to characterize susceptibility of any graft material to bacterial biofilm prior to implantation.

Carbohydrate receptors of bacterial adhesins: implications and reflections.

Bacteria entering a host depend on adhesins to achieve colonization. Adhesins are bacterial surface structures mediating binding to host surficial areas. Most adhesins are composed of one or several proteins. Usually a single bacterial strain is able to express various adhesins. The adhesion type expressed may influence host-, tissue or even cell tropism of Gram-negative and of Gram-positive bacteria. The binding of fimbrial as well as of afimbrial adhesins of Gram-negative bacteria to host carbohydrate structures (=receptors) has been elucidated in great detail. In contrast, in Gram-positives, most well studied adhesins bind to proteinaceous partners. Nevertheless, for both bacterial groups the binding of bacterial adhesins to eukaryotic carbohydrate receptors is essential for establishing colonization or infection. The characterization of this interaction down to the submolecular level provides the basis for strategies to interfere with this early step of infection which should lead to the prevention of subsequent disease. However, this goal will not be achieved easily because bacterial adherence is not a monocausal event but rather mediated by a variety of adhesins.

Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis.

Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

Alternative transcription factor sigma(B) is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate.

Osmotic stress was found to induce biofilm formation in a Staphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor sigma(B), resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding sigma(B) completely restored the wild-type phenotype. The combined data suggest a critical role of sigma(B) in S. aureus biofilm regulation under environmental stress conditions.

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus.

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Impact of flavophospholipol and vancomycin on conjugational transfer of vancomycin resistance plasmids.

The influence of vancomycin and flavophospholipol (FPL) on the transfer rate of conjugative plasmids harboring the vancomycin resistance operon vanA was determined in several clinical and animal isolates of Enterococcus faecium. FPL significantly inhibited the frequency of transfer of conjugative VanA plasmids up to 70-fold. Vancomycin had no significant effect on the transfer rate of VanA plasmids.

Regulation of sigmaB-dependent transcription of sigB and asp23 in two different Staphylococcus aureus strains.

The alkaline shock protein Asp23 was identified as a sigmaB-dependent protein in Staphylococcus aureus. In Bacillus subtilis, the asp23 promoter from S. aureus is regulated like other sigmaB-dependent promoters, which are strongly induced by heat and ethanol stress. However, almost no induction of asp23 expression was found after heat or ethanol stress in S. aureus MA13 grown in a synthetic medium, where the basal expression level of asp23 is high. Under the same experimental conditions the sigmaB gene itself showed a similar expression pattern: it was highly expressed in synthetic medium but not induced by heat or ethanol stress. In contrast, sigmaB activity was increased by heat stress when the cells were grown in a complex medium. The constitutive expression of sigB and sigmaB-dependent stress genes in S. aureus MA13 grown in a synthetic medium is in a sharp contrast to the regulation of sigmaB activity in B. subtilis, and needs further investigation. A deletion of 11 bp in the rsbU gene, which encodes the phosphatase that acts on RsbV (the anti-anti-sigma factor), in S. aureus NCTC 8325-4 might be responsible for the failure of heat stress to activate sigmaB in complex medium, and thus reduce the initiation of transcription at sigmaB-dependent promoters in this strain.

Evolution of bacterial pathogenesis.

The evolution of bacteria is associated with continuous generation of novel genetic variants. The major driving forces in this process are point mutations, genetic rearrangements, and horizontal gene transfer. A large number of human and animal bacterial pathogens have evolved the capacity to produce virulence factors that are directly involved in infection and disease. Additionally, many bacteria express resistance traits against antibiotics. Both virulence factors and resistance determinants are subject to intrastrain genetic and phenotypic variation. They are often encoded on unstable DNA regions. Thus, they can be readily transferred to bacteria of the same species or even to non-related prokaryotes. This review article focuses on the main mechanisms of bacterial microevolution responsible for the rapid emergence of variants with novel virulence and resistance properties. In addition, processes of macroevolution are described with special emphasis on gene transfer and fixation of adaptive mutations in the genome of pathogens.

Effects of subinhibitory concentrations of antibiotics on alpha-toxin (hla) gene expression of methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates.

Concentrations of antibiotics below the MIC are able to modulate the expression of virulence-associated genes. In this study, the influence of subinhibitory doses of 31 antibiotics on the expression of the gene encoding the staphylococcal alpha-toxin (hla), a major virulence factor of Staphylococcus aureus, was investigated with a novel gene fusion protocol. The most striking observation was a strong induction of hla expression by subinhibitory concentrations of beta-lactams and an almost complete inhibition of alpha-toxin expression by clindamycin. Whereas glycopeptide antibiotics had no effect, the macrolide erythromycin and several aminoglycosides reduced and fluoroquinolones slightly stimulated hla expression. Furthermore, Northern blot analysis of hla mRNA and Western blot (immunoblot) analysis of culture supernatants of both methicillin-sensitive and methicillin-resistant S. aureus strains revealed that methicillin-induced alpha-toxin expression is a common phenomenon of alpha-toxin-producing strains. Some methicillin-resistant S. aureus isolates produced up to 30-fold more alpha-toxin in the presence of 10 microg of methicillin per ml than in its absence. The results indicate that the novel gene fusion technique is a useful tool for studying the modulation of virulence gene expression by antibiotics. Moreover, the results suggest that the effects of certain antibiotics on virulence properties may be relevant for the management of S. aureus infections.

A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis.

The SpoOJA and SpoOJB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spoOJB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of SpoOJA activity in the absence of SpoOJB, and its basis was unknown. In the studies reported here, SpoOJA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor SpoOA and RNA polymerase with the spollG promoter. This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo. SpoOJB is known to neutralize SpoOJA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning. These data suggest that SpoOJA and SpoOJB form a regulatory link between chromosome partition and development gene expression.

Analysis of expression of the alpha-toxin gene (hla) of Staphylococcus aureus by using a chromosomally encoded hla::lacZ gene fusion.

The staphylococcal alpha-toxin (Hla) is a major virulence factor contributing to Staphylococcus aureus pathogenesis. To elucidate the conditions influencing hla expression, the determinant was fused to lacZ, the reporter gene coding for beta-galactosidase. The hla::lacZ fusion was integrated into the chromosome of the wild-type S. aureus strain Wood 46, leading to the variant Wood 46-3. Alpha-toxin expression was found to be dependent on temperature, showing a maximum at 42 degrees C. Furthermore, the indicator strain showed a growth phase-dependent hla regulation which was influenced by temperature. At 37 degrees C, induction of hla::lacZ expression occurred in the late exponential phase of growth, whereas at 42 degrees C, a strong induction was observed as early as the mid-exponential phase. These observations were verified by Northern blot analysis of hla mRNA and by Western blot (immunoblot) analysis of culture supernatants of strain Wood 46. It was additionally found that the induction of hla transcription at 42 degrees C was not coupled with higher concentrations of agr RNAIII, the effector molecule of the global regulator agr. Furthermore, expression of the alpha-toxin was repressed at a high osmolarity. It was also shown that oxygen is essential for hla expression and that cultivation of the S. aureus strain Wood 46-3 on solid medium and in the presence of carbon dioxide stimulated hla transcriptional activity.

Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning.

The Bacillus subtilis spo0J gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spo0J-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for division. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria.

[Parenthood and child development after reproduction medicine treatment. Psychosomatic follow-up of 46 parents after assisted reproduction]. / Elternschaft und kindliche Entwicklung nach reproduktionsmedizinischer Behandlung. Psychosomatischem Begleitung und Evaluation bei 46 Elternpaaren nach assistierter Reproduktion.

On the basis of a longitudinal study on "parenthood and child development after successful conception by IVF" in which 46 couples with 64 children born after reproductive medical treatment (IVF or ICSI) were followed up for three years, we now discuss several aspects of the psychological situation of those involved. Reference is made to the characteristics of the "structure" of the couples, their attitude towards the treatment, their experience of the pregnancy, features of child development and of the parent-child relationship, as well as to the prevailing psychological strategies of the couples for coping with the stresses and risks of assisted fertilization. Preliminary results show that during the course of treatment and following the birth of the children, the partnerships remained stable, and that relationships between family members are, for the most part, developing favorably.

Deactivation of the sporulation transcription factor Spo0A by the Spo0E protein phosphatase.

The spo0E locus of Bacillus subtilis codes for a negative regulator of sporulation that, when overproduced, represses sporulation and, if deleted, results in inappropriate timing of sporulation. The product of this locus, Spo0E, was purified and found to be a protein phosphatase, which specifically dephosphorylated the sporulation transcription factor Spo0A-P, converting it to an inactive form. Spo0E was not significantly active as a phosphatase on other components of the phosphorelay signal-transduction pathway producing Spo0A-P. A mutant Spo0E protein that results in sporulation deficiency was purified and found to be hyperactive as a phosphatase. The Spo0E phosphatase may provide an additional control point for environmental, metabolic, or cell-cycle regulation of phosphate flow in the phosphorelay. These results reinforce the concept that the phosphorelay is subject to a host of positive and negative signals for sporulation that are recognized and interpreted as signal integration circuit that has the role of regulating the cellular level of active phosphorylated Spo0A sporulation transcription factor.

Melting during steady-state transcription of the rrnB P1 promoter in vivo and in vitro.

The rRNA rrnB P1 promoter was probed with the single-strand-selective reagent potassium permanganate during steady-state transcription in vitro and in vivo. In both cases, a weak but significant level of permanganate sensitivity was observed, which was not changed by treatment with rifampin. In contrast, static studies showed that rifampin strongly affects the very high level signal associated with polymerases that have used ATP and CTP as initiating nucleotides. We infer that the permanganate sensitivity associated with steady-state transcription is due to polymerases that have not yet used ATP and CTP. The slow and regulated step during rrnB P1 transcription may be the use of the initiating nucleotides to catalyze stable opening of the promoter DNA.

DNA melting within stable closed complexes at the Escherichia coli rrnB P1 promoter.

Several transcription complexes are shown to form on the E. coli ribosomal rrnB P1 promoter in vitro. These include two closed complexes that are sensitive to heparin attack, and one open complex. The closed complexes are unusual in that they are both highly specific and stable, properties associated with the atypical DNA sequence of this promoter. The effector ppGpp does not prevent closed complex formation but does reduce the level of open complexes that form.

Interrelated effects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter.

The formation of complexes containing high levels of DNA melting at the ribosomal RNA rrnB P1 promoter in vitro is shown to be facilitated by DNA supercoiling or low salt. The effector nucleotide ppGpp is ineffective under these conditions. The loss of supercoils or addition of salt increases the effectiveness of ppGpp in inhibiting formation of these complexes. In vivo plasmid DNA supercoiling is shown to decrease during starvation protocols that also increase levels of ppGpp. The results suggest that ppGpp regulation may be affected by the state of DNA supercoiling in vivo.

Cochlear blood flow in response to dilating agents.

Reduced cochlear blood flow (CBF) has been implicated in various pathologies of the inner ear, including sudden deafness, noise-induced hearing loss and Meniere's disease. Thus the aim of some current therapeutic regimens to treat these conditions is to increase CBF and thereby improve oxygenation of the inner ear tissues. Most of the vasodilating agents in clinical use, however, do not have specific experimental evidence to support their effects on CBF. The hypotension which can follow systemic administration may limit their local effectiveness and general utility, just as it complicates the interpretation of the data in animal experiments. In the current study we investigated the effect of six agents, known for their systemic cardiovascular actions, on CBF: hydralazine, sodium nitroprusside, papaverine, nicotinic acid, verapamil and histamine. The effect of these drugs was studied after topical applications on the round window membrane (RWM) and systemic intravenous administrations. CBF was monitored with a laser Doppler flowmeter (LDF). Topical administration of sodium nitroprusside was the most effective in increasing CBF, followed, in order, by hydralazine and histamine. No change in CBF was observed for papaverine, verapamil or nicotinic acid. Systemic administrations of all the agents caused a marked decrease in blood pressure and variable effects on CBF. We discuss the CBF changes in relation to the different pharmacological mechanisms of action of each drug. The study demonstrates the effectiveness of topical application of vasodilating agents in increasing CBF.