The effect of a semisynthetic pentosan-polysulfo-ester (SP 54) upon the complement system in vitro has been studied in comparison with the effects of heparin. Both substances inhibit in small concentrations the Cl-esterase. There are differences in the effect of SP 54 and heparin. According to the in vitro results SP 54 seems to have a ten times higher anticomplementary action. At the same time the influence upon coagulation is less. SP 54 appears to be a substance of therapeutic interest.

A 48-year-old woman required mechanical ventilation after aortic valve replacement for decompensated aortic valve stenosis when bleeding complications developed and rethoracotomy had to be performed. Acute renal failure necessitated haemodialysis. Septic fever of unknown aetiology failed to respond to oxacillin, cefotaxim and tobramycin. The endotracheal cannula and central venous catheter were changed on the 24th postoperative day and the antibiotic treatment altered to 250 mg imipenem and 125 mg vancomycin three times daily intravenously. The fever soon subsided, but recurred on the 32nd postoperative day, accompanied by increasing leucocytosis. The patient was obstipated but had no intraabdominal signs. Four days later ultrasonography demonstrated thickening of the intestinal wall and coloscopy showed typical pseudomembranous colitis. Intestinal contents were positive for Clostridium difficile toxin. Despite immediate rectal and intragastric administration of 250 mg vancomycin four times daily the patient died of pseudomembranous colitis, confirmed at autopsy. The case demonstrates that vancomycin cannot always prevent the development of pseudomembranous colitis.

ABSTRACT A putative pullulanase-encoding gene from Streptococcus pneumoniae was identified by screening a genomic expression library with human convalescent-phase serum. The 3,864-bp gene encoded a 143-kDa protein. Surface location and pullulanase activity of the protein, designated SpuA, was demonstrated. SpuA was present in all investigated pneumococcal isolates of different serotypes. The spuA 5′ end was highly conserved among clinical isolates except for a 75-bp region. The properties of SpuA reported here indicate that this novel immunogenic surface protein might have potential as a vaccine target.

The purpose of the study was to analyze the distribution of the macrolide-resistance genes in 134 erythromycin-resistant Staphylococcus aureus blood-culture isolates collected at 15 German university hospitals. The most prevalent resistance gene was ermC (68/134; 50.7%), followed by ermA (52/134; 38.8%), ereB (10/134; 7.5%), and mrsA/msrB (4/134; 6%). The least common genes were ermB (3/134; 2.2%) and ereA (1/134; 0.7%). Overall, resistance to erythromycin was predominantly due to the presence of two erm genes, although with different distributions, depending on the methicillin-resistance pattern.

The most prominent gamma interferon (IFN-gamma)-induced antimicrobial effector mechanisms are the induction of nitric oxide (NO) synthase (NOS) and of indoleamine 2,3-dioxygenase (IDO) activity. We have recently found that human glioblastoma cells and human macrophages inhibit the growth of group B streptococci after stimulation with IFN-gamma. In this report, we show that in addition, human RT4 (uroepithelial) cells can inhibit the growth of enterococci. Murine macrophages (RAW cells) are unable to inhibit bacterial growth after IFN-gamma stimulation. Stimulation of human glioblastoma cells, macrophages, and RT4 cells with human IFN-gamma results in a strong expression of IDO activity; however, NO production remains undetectable. In strong contrast, murine RAW cells produce large amounts of NO when stimulated with murine IFN-gamma and IDO activity is not detectable. Interleukin-1 (IL-1) induces NO synthase in human RT4 cells when the cells are costimulated with IFN-gamma. We found that IL-1 inhibits IFN-gamma-stimulated IDO activity and antimicrobial effects in RT4 cells, while in human glioblastoma cells, which lack detectable NO synthase activity, neither of these effects was altered by costimulation with IFN-gamma and IL-1. The IL-1-mediated inhibition of IDO activity and of subsequent antibacterial effect is due to the production of NO. This conclusion was supported by evidence that N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible NOS activity, is able to block the inhibitory action of IL-1 on IFN-gamma-induced bacteriostasis. We therefore conclude that NO production does not inhibit the growth of enterococci but might be involved in the regulation of IDO activity in some human cells.

Mycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100). To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene. With this strategy, we identified a genomic region of about 10. 4 kb in M. hominis FBG carrying the P100 gene. Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region. Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized. The organization of the P100 gene and oppBCDF in a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb. Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene. The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system in M. hominis.

Recombinant peptides of Mycoplasma hominis adhesin P50 were expressed in Escherichia coli to investigate which regions of the P50 molecule are responsible for cyto-adherence. The respective DNA fragments were obtained by PCR amplification of the p50 gene with the use of mutating oligonucleotides to change the TGA codons of mycoplasma to TGG codons, which are translated in E. coli as tryptophan. The resulting three clones (I, I+II and I+III) contained regions of P50 which closely represent the repeat regions A, A+B and A+C. After expression in E. coli, the polyhistidine-tagged recombinant peptides were purified by metal chelation chromatography. The three recombinant peptides were detected in Western blot analysis by a polyclonal antiserum directed against M. hominis FBG and two P50-specific monoclonal antibodies, BA10 and BG2. Each of the three recombinant peptides I, I+II and I+III was able to adhere to immobilised HeLa cells in an adhesion assay. The cyto-adhesion of the peptides could be inhibited by pre-incubation with the appropriate antibody. Therefore, it is suggested that adherence may be mediated by all regions of the P50 molecule. Attachment of the recombinant peptides to immobilised HeLa cells was inhibited by high mol. wt dextran sulphate (MW 500000), indicating that the respective P50 regions bind to sulphatides on the host cell membrane.

The influence of the intracellular parasite Toxoplasma gondii on macrophage expression of co-stimulatory molecules was studied. Unlike surface expression of CD80/B7-1, that of CD86/B7-2 is increased in mouse peritoneal macrophages 24 h following exposure to live toxoplasma in vitro. Most CD86 molecules are found on infected cells bearing a maximum parasite load. Consistent with the elevated membrane expression, the quantity of CD86 gene transcript is increased in macrophages infected by T. gondii in vitro or in vivo. CD86 up-regulation contributes to the augmented capacity of parasitized macrophages to present antigen to tuberculin-specific CD4+ T cells as demonstrated by blocking CD86 ligand interaction. T. gondii triggers up-regulation of CD86 in macrophages from BALB/c mice which are resistant to the development of toxoplasmic encephalitis. Infection of macrophages from the susceptible strain BALB.B, however, results in a decreased surface expression of CD86, although the parasite load and intracellular proliferation proved comparable in both macrophages. This differential host cell reaction correlates with disparate profiles in T. gondii-induced cytokine secretion. Upon challenge with toxoplasma, IL-1alpha and tumor necrosis factor (TNF)-alpha are released to a significantly higher extent by BALB/c than by BALB.B macrophages, whereas the latter secrete more IL-12 and IL-10. In BALB.B macrophages, T. gondii-induced IL-10 down-regulates surface expression of CD86, thus indicating an interference of parasite-dependent cytokine release and modulation of CD86. The biased secretory response in macrophages from the two congenic strains implies an MHC-dependent and dichotomous monokine induction by T. gondii. Up-regulation of CD86 seems to occur along the IL-1/TNF-inducing pathway and experimental evidence indicates that this enhances T cell activation by parasitized macrophages.

Toxoplasma gondii, an obligate intracellular parasite. is able to replicate in human brain cells. We recently showed that interferon (IFN)-gamma-activated cells from glioblastoma line 86HG39 were able to restrict Toxoplasma growth. The effector mechanism responsible for this toxoplasmostatic effect was shown by us to be the IFN-gamma-mediated activation of indolamine 2.3-dioxygenase (IDO). resulting in the degradation of the essential amino acid tryptophan. In contrast. glioblastoma 87HG31 was unable to restrict Toxoplasma growth after IFN-gamma activation. and IFN-gamma-mediated IDO activation was weak. We observed that tumor necrosis factor (TNF)-alpha alone is unable to activate IDO or to induce toxoplasmostatic in any glioblastoma cell line tested. Interestingly, we found that TNF-alpha and IFN-gamma were synergistic in the activation of IDO in glioblastoma cells 87HG31. 86HG39 and U373MG and in native astrocytes. This was shown by the measurement of enzyme activity as well as by the detection of IDO mRNA in TNF-alpha + IFN-gamma activated cells. This IDO activity results in a strong toxoplasmostatic effect mediated by glioblastoma cells activated simultaneously by both cytokines. Antibodies directed against TNF-alpha or IFN-gamma were able to inhibit IDO activity as well as the induction of toxoplasmostasis in glioblastoma cells stimulated with both cytokines. Furthermore. it was found that the addition of L-tryptophan to the culture medium completely blocks the antiparasitic effect. We therefore conclude that both TNF-alpha and IFN-gamma may be involved in the defense against cerebral toxoplasmosis by inducing IDO activity as an antiparasitic effector mechanism in brain cells.

As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.

Neonatal sepsis is most often caused by group B streptococci (GBS) and is a major cause of death in the neonatal period. The response of the immune system in the newborn child has received much attention and is thought to be deficient in a number of ways. The effector response of neonatal monocyte-derived macrophages (MDM) was investigated. Interferon-gamma induced the activation of indoleamine 2,3-dioxygenase in MDM and inhibited the growth of GBS. Both effects were enhanced by the addition of tumor necrosis factor-alpha to the culture conditions. The coincident supplementation of L-tryptophan with the bacteria abrogated the bacterial growth inhibition, thus confirming the causative role of L-tryptophan depletion. Control of the extracellular as well as intracellular L-tryptophan levels may thus be one of the effector mechanisms with which the immune system defends the host against GBS dissemination and disease.

The clp60 gene encoding P60, a conserved lipoprotein of Mycoplasma hominis, was cloned and sequenced from both the type strain PG21 and the isolate FBG. Both open reading frames were identical in length, comprising 1746 nucleotides. The deduced amino acid sequences differed in 16 out of 582 amino acids. As expected, none of these divergences mapped within the epitope that was recognized by mAb CG4 in all of the 198 isolates of M. hominis analyzed so far. This conserved epitope was narrowed down to amino acids 454 through 464 within the C terminus of P60. For the expression of the recombinant homolog P60, P60rec, in E. coli the TGA codons of clp60 were substituted for TGG codons prior to cloning of clp60 into the expression plasmid pQE41. The expression of P60rec as a fusion protein with dihydrofolate reductase carrying an N-terminal His-tag enabled the purification of large amounts of P60rec in a soluble form.

A characteristic feature of the mycoplasmas is the presence of variable surface proteins which may play an important role in the adaptation of the cell-wall-less organisms to their host environments. In addition, this antigen variation may be an important pathogenic property of the organism. The ubiquity of the gene encoding P50, an adhesin of Mycoplasma hominis FBG, and its transcription were analysed in different isolates of M. hominis. The p50 gene was present in all isolates tested. Based on Southern blot analysis and sequencing of the gene, the isolates could be classified into one of three distinct groups. Within two groups specific truncations of the p50 gene occurred. The reduction of the gene size was confirmed in Northern blot analysis of representative isolates from each group, with a decrease in transcript length from 1.6 kb in group G-1 down to 0.76 kb in group G-3. In addition to truncation, a coincidental duplication of some gene segments was detected. This work has provided evidence for the genetic basis of a further variation in the M. hominis P50 adhesin.

One hundred sixteen unrelated clinical isolates of Staphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the presence of mutations in the grlA, grlB, gyrA, and gyrB gene loci. Two mutations within grlA (located at codons 80 and 84) and two mutations within gyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.

Whole bacterial cells of Staphylococcus aureus as well as purified staphylococcal peptidoglycan (PG) have been demonstrated to stimulate human monocytes to release cytokines. Hypothesising that the phenomenon of changes induced by beta-lactam antibiotics in cell-wall composition may alter the immunological properties of the intact cell wall as well as of purified cell-wall components, this study assessed whether cytokine release by human monocytes is altered if cells from strains grown in the presence or absence of sub-minimal inhibitory concentrations of oxacillin are used as stimuli. Whole bacterial cells and isolated PG from these strains, grown in the presence of oxacillin, showed a significantly reduced stimulation of tumour necrosis factor-alpha, interleukin (IL)-1 beta and IL-6 release by human monocytes in a concentration-dependent fashion. The serum-induced potentiation of cytokine production by human monocytes in response to PG with modified cross-linking was also reduced. These observations may have particular relevance for staphylococcal infections, in which clinically achievable beta-lactam concentrations do not suppress staphylococcal growth yet may alter and possibly enhance virulence.

In Staphylococcus aureus, in addition to mutations in the grl and gyr gene loci, multidrug efflux pumps like NorA contribute to decreased fluoroquinolone susceptibility. Efflux pumps can be inhibited by the plant alkaloid reserpine, which, at 20 mg/L, reduced sparfloxacin, moxifloxacin and ciprofloxacin IC(50)s and MICs by up to four-fold in 11, 21 and 48 of the 102 unrelated clinical isolates tested, respectively. The effect was less pronounced with the hydrophobic drugs sparfloxacin and moxifloxacin than with the hydrophilic drug ciprofloxacin and was stable in all 25 clonally related isolates tested.

Zusammenfassung: Nosokomiale Infektionen durch Methicillin-resistente Staphylococcus aureus-Stämme (MRSA) stellen in Krankenhäusern und dort besonders auf Intensivstationen ein wachsendes Problem dar.Aus diesem Grund ist der Bedarf an Typisierungsverfahren, die schnell und