Antigenicity, expression, and molecular characterization of surface-located pullulanase of Streptococcus pneumoniae.

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.

Distribution of macrolide-resistance genes in Staphylococcus aureus blood-culture isolates from fifteen German university hospitals. M.A.R.S. Study Group. Multicentre Study on Antibiotic Resistance in Staphylococci.

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.

Interleukin-1 inhibits gamma interferon-induced bacteriostasis in human uroepithelial cells.

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.

The adherence-associated lipoprotein P100, encoded by an opp operon structure, functions as the oligopeptide-binding domain OppA of a putative oligopeptide transport system in Mycoplasma hominis.

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.

Differential CD86/B7-2 expression and cytokine secretion induced by Toxoplasma gondii in macrophages from resistant or susceptible BALB H-2 congenic mice.

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.

Inducible anti-parasitic effector mechanisms in human uroepithelial cells: tryptophan degradation vs. NO production.

In murine cells the most important effector mechanism directed against the intracellular pathogen Toxoplasma gondii is the production of toxic nitrogen oxides. In contrast the induction of the tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO) has been described to be the most effective anti-parasitic mechanism in most human cells. In this report we analysed IDO induction and NO production in the human uroepithelial carcinoma cell line RT4. We found that after stimulation with IFN-gamma these cells were able to restrict toxoplasma growth. This was due to an activation of IDO, and the anti-parasitic effect mediated by RT4 cells was abrogated by the addition of L-tryptophan. In addition we found that the costimulation of RT4 cells with IL-1 and IFN-gamma results in the production of nitric oxide, and that in RT4 cells stimulated with both these cytokines, IDO activity and toxoplasmostasis was lower than in cells stimulated with IFN-gamma alone. This IL-1-mediated inhibition of IFN-gamma-induced IDO activity and toxoplasmostasis could be blocked by monomethyl L-arginine, an inhibitor of NO production. We therefore conclude that the induction of indolamine 2,3-dioxygenase activity in human cells is a very important effector mechanism directed against Toxoplasma gondii, and that in human cells the production of NO might be involved in the regulation of IDO activity.

Cloning and expression of P60, a conserved surface-localized protein of Mycoplasma hominis, in Escherichia coli.

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.

Interferon-gamma-induced activation of indoleamine 2,3-dioxygenase in cord blood monocyte-derived macrophages inhibits the growth of group B streptococci.

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.

Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC.

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.

Development of a multiplex-PCR for direct detection of the genes for enterotoxin B and C, and toxic shock syndrome toxin-1 in Staphylococcus aureus isolates.

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.

Reduction in cytokine release from human monocytes by modifications in cell wall structure of Staphylococcus aureus induced by subinhibitory concentrations of oxacillin.

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)-1beta 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.

The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus.

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 IC50s 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.

Enterotoxin and toxic shock syndrome toxin-1 production of methicillin resistant and methicillin sensitive Staphylococcus aureus strains.

In this study the production of enterotoxin A-D and toxic shock syndrome toxin-1 (TSST-1) of 181 methicillin resistant (MRSA) and 100 methicillin sensitive (MSSA) Staphylococcus aureus first isolates from different patients was investigated. All the MRSA- and MSSA isolates in the study were collected in a period between 1993 and 1995 from specimens sent from 11 different acute care hospitals in the greater Düsseldorf area. As far as possible the isolates were matched according to ward and hospital. The isolates were collected in the same time period and matched for specimen from which isolated. Furthermore, only first isolates were analysed in both groups. No significant difference in the production of toxin of any type between MRSA and MSSA could be detected (51 and 40% respectively). When the individual toxins were analysed, again no significant difference between MRSA and MSSA was demonstrable (enterotoxin production by MRSA 40% and MSSA 36%, and TSST-1 16% and 8% respectively). Despite this, a slight tendency for MRSA to produce enterotoxin A and B and for MSSA to produce enterotoxin C was observed. In addition, generation of TSST-1 by both groups was independent of enterotoxin A-D production. Interestingly, no increase in the proportion of TSST-1- or enterotoxin-producing MRSA and MSSA isolates was observed in strains isolated from blood cultures from patients with a clinical diagnosis of sepsis. Genotypical pulsed-field-gel-electrophoresis (PFGE) and phenotypical (bacteriophage typing, lysotyping) characterization of the 181 MRSA isolates resulted in 28 different PFGE patterns (of which 19 were toxin producers) and 22 lysotyping groups (18 of which produced toxin). In summary, the investigated clinical S. aureus isolates showed no difference in their ability to produce toxin and this was independent of their sensitivity to methicillin.

Methicillin resistant Staphylococcus aureus strains in the greater Düsseldorf area.

Over a period of three years the incidence of methicillin resistant Staphylococcus aureus (MRSA) isolates in 11 hospitals in the greater Düsseldorf area was observed. From a total of 7,814 S. aureus isolates, 489 (6.3%) were methicillin resistant. From 198 different patients, MRSA first isolates and 291 second isolates could be cultured. Methicillin resistance among all S. aureus isolates from 11 hospitals in the greater Düsseldorf area, ranged from 0.5 to 7.8% dependant on the size of the hospital. The highest incidence (7.8%) was found in a 1,500 bed hospital and the lowest incidence in a smaller 200 bed hospital (0.5%). With respect to the distribution among clinical departments the highest incidence of MRSA isolates was found on intensive care units and surgical wards, 25.5% and 13.0% respectively. The commonest specimen from which the MRSA isolates were cultured were respiratory secretions (17.6%) followed by central venous catheter tips (12.8%). In terms of the drug resistance pattern: all isolates were resistant to the aminoglycosides and gyrase inhibitors, whereas between 80% and 90% were sensitive to fusidic acid, chloramphenicol and pyrimethamine-sulfamethoxazole. All the strains were sensitive to the glycopeptide antibiotics, vancomycin and teicoplanin. Strain typing of 181 available first isolates (from a total of 198 first isolates) by PFGE and phage lysotyping produced identical results in more than 90% of all cases. Twenty-eight different MRSA strain types were identified by PFGE and in total 23 lysotypes could be determined. During the period of investigation an increased incidence of MRSA on an intensive care unit was observed, in which a total of 204 MRSA (42% of the total number) were isolated. The strain typing using both methods showed that on that ICU eight different MRSA types were involved in this outbreak. A hygiene plan was implemented on the unit with considerable success in reducing the incidence and spread of MRSA.

[Insertion possibility of 16S-23S space amplification and random amplified polymorphic DNA analysis for typing of methicillin-resistant Staphylococcus aureus strains in the context of nosocomial infections]. / Einsatzmöglichkeiten der 16S-23S-Spacer-Amplifikation und der "Random Amplified Polymorphic DNA"--Analyse zur Typisierung Methicillin-resistenter Staphylococcus aureus-Stämme im Rahmen nosokomialer Infektionen.

Within the scope of the present study n = 183 MRSA isolates from the extended area of Düsseldorf and n = 93 international MRSA strains from seven different countries were typed by pulsed-field gel electrophoresis and two PCR methods (RAPD and 16S-23S-spacer amplification). The isolates could be subdivided into 30 different types by PFGE, into 21 by means of RAPD and 18 by 16S-23S-spacer amplification. PFGE had the highest discriminatory potential, however, a combined use of the three typing methods allows a more detailed differentiation even of those isolates with identical PFGE pattern. Both amplification procedures were rapid, easy in handling with reproductable results. For a temporary epidemiological analysis within 24 hours, both amplification methods could be combined. In case the investigated isolates were still suspected of showing a "clonal identity", they should be analysed by additional PFGE (lasting about four days). Although the international isolates were chosen by random selection, several MRSA strains with identical pattern could be found in different countries of the world. Some RAPD-, spacer- and PFGE pattern were constant over many years. This reflects a high genetic stability of single strains.

Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells.

To investigate the role of astroglia in intracerebral immune response to Toxoplasma gondii, astrocytes cultured from mouse brain were inoculated with mouse-virulent or -avirulent toxoplasma strains. In comparison to microglia/ brain macrophages, astrocytes as host cells allowed stronger proliferation of avirulent parasites. Toxoplasma infection of astroglia was accompanied by release of interleukin- (IL)1 alpha, IL-6, and granulocyte/macrophage colony-stimulating factor (GM-CSF) activity, whereas alternative challenge by lipopolysaccharide (LPS) evoked no IL-1 response and significantly higher titers of IL-6 and GM-CSF. At the mRNA level, both stimuli induced transcription of all three cytokines in astrocytes. Secretion of IL-1 and IL-6 upon infection was triggered by T. gondii brady- and tachyzoites in a time- and dose-dependent manner. Heat killing of parasites, but not an exposure to polymyxin B, abrogated their cytokine-inducing activity, thus indicating that an LPS-independent stimulus is provided by T. gondii. When administered in combination, LPS synergistically augmented the IL-1-inducing effect of toxoplasma infection. In comparison, T. gondii-induced, but not an LPS-triggered, IL-6 response of astrocytes resisted to antagonization with IL-10. The IL-6 response of parasitized astroglia was up-regulated by external tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta 1, with only TNF-alpha enhancing simultaneous release of IL-1. Substantial secretion of IL-10 and TNF-alpha was detected in T. gondii-infected microglia, but not in astrocyte cultures. A possibly autocrine stimulation of infected astroglia via IL-1 was found to be unlikely, since addition of IL-1 receptor antagonist did not affect the release of IL-6 and GM-CSF while inhibiting these responses in IL-1-treated cells. The findings substantiate a separate, T. gondii-induced pathway of astroglia activation characterized by the release of IL-1 which may drive local inflammatory reaction both at initial infection of the brain and during reactivating toxoplasmosis.

Cellular immune reactions directed against Toxoplasma gondii with special emphasis on the central nervous system.

Toxoplasma gondii is an obligate intracellular parasite which, after primary infection of humans, is maintained in a dormant state by the host cellular immune system. In the event of an acquired immunosuppression, those parasites surviving as dormant cysts in the host may undergo a change in status, proliferate and cause a life-threatening toxoplasmic encephalitis. Over the last decade much knowledge has accumulated concerning the immune response against T. gondii. This review focuses attention particularly on the anti-parasitic effector mechanisms and the cellular immune reactions in the central nervous system during the course of reactivated toxoplasmic encephalitis.

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain.

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2+ neurons, GFAP+ astrocytes, F4/80+ microglia, and O1+ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in] parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection.

Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.