Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1ß, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

Intracellular proliferation of S. aureus in osteoblasts and effects of rifampicin and gentamicin on S. aureus intracellular proliferation and survival.

Staphylococcus aureus is the most clinically relevant pathogen regarding implant-associated bone infection and its capability to invade osteoblasts is well known. The aim of this study was to investigate firstly whether S. aureus is not only able to invade but also to proliferate within osteoblasts, secondly to delineate the mechanism of invasion and thirdly to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival of S. aureus. The SAOS-2 osteoblast-like cell line and human primary osteoblasts were infected with S. aureus EDCC5055 and S. aureus Rosenbach 1884. Both S. aureus strains were able to invade efficiently and to proliferate within human osteoblasts. Immunofluorescence microscopy showed intracellular invasion of S. aureus and transmission electron microscopy images could demonstrate bacterial division as a sign of intracellular proliferation as well as cytosolic bacterial persistence. Cytochalasin D, the major actin depolymerisation agent, was able to significantly reduce S. aureus invasion, suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 µg/mL of rifampicin was able to inhibit bacterial survival in SAOS-2 cells with almost complete elimination of bacteria after 4 h. Gentamicin could also kill intracellular S. aureus in a dose-dependent manner, an effect that was significantly lower than that observed using rifampicin. In conclusion, S. aureus is not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication of S. aureus whereas gentamicin only poorly eliminates intracellularly replicating bacteria.

Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX-M-15 in a tertiary hospital in Tanzania.

The molecular epidemiology of 32 non-duplicate, CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains, isolated from clinical samples, was investigated. Multilocus sequence typing revealed multiple sequence type clonal complexes: ST131 (12), ST405 (4), ST638 (3), ST38 (2), ST827 (2), ST224 (1), ST648 (1), ST46 (1) and two new sequence type clonal complexes (1845 and 1848) in 22 pulsed field gel electrophoresis clusters. The bla(CTX-M-15) gene was located on conjugative IncF plasmids. This is the first report of the worldwide emerging clonal complex ST131 linked to bla(CTX-M-15) in Tanzania and demonstrates the need for constant surveillance in developing countries to prevent the spread of these multiresistant isolates.

[Update: invasive fungal infections: Diagnosis and treatment in surgical intensive care medicine]. / Update: invasive Pilzinfektionen: Diagnose und Therapie in der operativen Intensivmedizin.

Fungal infections are of great relevance in surgical intensive care and Candida species represent the predominant part of fungal pathogens. Invasive aspergillosis is also relevant especially in patients with chronic pulmonary diseases. It is crucial for therapy success to begin adequate antifungal treatment at an early stage of the disease. Risk stratification of individual patient symptoms is essential for therapy timing. In case of suspected or proven candida infection, fluconazole is the agent of choice when the patient is clinically stable and no azoles have been administrated in advance and the local epidemiology makes azol resistance unlikely. For clinically instable patients with organ dysfunction the echinocandins serve as primary therapy because of their broad spectrum and reasonable safety profile. Due to a relevant proportion of azole resistant Candida species, susceptibility testing should be done routinely. Depending on the species detected de-escalating to an azole is feasible if organ dysfunctions have resolved. An invasive aspergillosis is primarily treated with voriconazole.

Hidden pathogens uncovered: metagenomic analysis of urinary tract infections.

Urinary tract infections (UTIs) are the most common kidney and urologic diseases in industrial nations and are usually caused through faecal contamination of the urinary tract. In this study, we have examined 1449 urine specimens both by culture and by PCR. The majority of UTIs examined were caused by Escherichia coli (35.15%), followed by miscellaneous bacteria (23.03%), and by Enterococcus faecalis (19.39%). A large fraction of fastidious and anaerobic bacteria (22.43%) was not detected under culture conditions but only by using PCR. This group of bacteria evade the standard culture conditions used in routine diagnostic laboratories examining urine specimens. The molecular approach used broad-range 16S rDNA PCR, denaturing high-performance liquid chromatography analysis, sequencing, and bioinformatic analysis to uncover these 'hidden' pathogens and is recommended in particular when examining leukocyte esterase-positive and culture-negative urinary tract specimens.

[Nanoparticulate silver. A new antimicrobial substance for bone cement]. / Nanopartikuläres Silber. Ein neues antimikrobielles Agens für Knochenzement.

BACKGROUND: Multiresistant bacteria have become an important problem in prosthetic joint infections. Their frequent resistance against gentamicin, which is commonly used in antibiotic-loaded bone cements, makes a new prophylaxis necessary. METHODS: PMMA-cement was loaded with 1% nanoparticulate silver and its antibacterial activity tested in vitro against gentamicin-resistant MRSE and MRSA strains as well as being compared to the activity of plain and gentamicin-loaded bone cements. A quantitative elution testing was also done to study the potentially cytotoxic effects of NanoSilver cement. RESULTS: Unloaded and PMMA-cement loaded with 2% gentamicin did not exhibit any antibacterial activity against MRSE and MRSA. At 1%, NanoSilver cement completely inhibited the proliferation of MRSA and MRSE. NanoSilver bone cement did not show any significant differences compared to the non-toxic control group. CONCLUSIONS: If these promising in vitro results can be confirmed in vivo, NanoSilver bone cement may be of considerable value in total joint arthroplasty.

Clonal identity of Candida albicans in the oral cavity and the gastrointestinal tract of pre-school children.

The clonal relationship between oral and fecal Candida albicans isolated from children of pre-school age was examined using RAPD analysis. Significantly higher levels of C. albicans were found in saliva, dental plaque, carious specimens and stools of 56 patients with severe caries as compared to 52 healthy control subjects. The highest prevalence was found in carious specimens and a strong correlation was observed between its presence in saliva, dental plaque, carious specimen and feces. RAPD analysis of isolates from 23 patients with simultaneous oral and fecal C. albicans revealed clonal counterparts present in both oral and stool samples in 15 cases; five patients harbored closely related strains; and three patients harbored unrelated strains. Our results demonstrate a strong correlation between oral and gastrointestinal C. albicans colonization. We assume that carious teeth may constitute an ecologic niche for C. albicans potentially responsible for recurrent oral and non-oral candidiasis.

Comparative genomics of Listeria species.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Bioinformatics in medical practice: what is necessary for a hospital?

Building bioinformatic facilities for a university hospital is pretty similar to using standardized building blocks to construct a house. Starting with the intention to built a dwelling house, a factory or just a shelter the architect draws a construction plan and determines the material to be used. In general, the building is then constructed by the workmen following exactly the plan. However, for particular reasons, minor alterations may be needed to improve the construction of the building. Here we use the metaphor of constructing a "bio-informatics building" to describe the steps needed to support the daily tasks of a university hospital medical microbiology department which uses genomic methods quite extensively for pathogen identification. Today the Giessen "bioinformatics building" is not yet complete but we have been able to lay solid foundations and erect the ground floor which is functional already. Using a combination of standard tools, internet accessible genomic databases and some own software tools we can support genome sequencing from the raw sequence to pathogen identification.

Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain.

Listeria monocytogenes is an opportunistic, food-borne human and animal pathogen. Host cell invasion requires the action of the internalins A (InlA) and B (InlB), which are members of a family of listerial cell-surface proteins. Common to these proteins are three distinctive N-terminal domains that have been shown to direct host cell-specific invasion for InlA and InlB. Here, we present the high-resolution crystal structures of these domains present in InlB and InlH, and show that they constitute a single "internalin domain". In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin (Ig)-like fold. The extended beta-sheet, resulting from the distinctive fusion of the LRR and the Ig-like folds, constitutes an adaptable concave interaction surface, which we propose is responsible for the specific recognition of the host cellular binding partners during infection.

Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells.

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria-mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.

Human endothelial cell activation and mediator release in response to Listeria monocytogenes virulence factors.

The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca(2+) suggested LLO-elicited Ca(2+) flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholipase C (EGD plcA mutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-kappa B to the stimulation with the LLO-producing strains EGD and Listeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-kappa B activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-kappa B translocation and the upregulation of cytokine synthesis. We conclude that L. monocytogenes is a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca(2+) flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-kappa B are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathogenic sequelae in severe listerial infection and sepsis.

Human dendritic cells infected by Listeria monocytogenes: induction of maturation, requirements for phagolysosomal escape and antigen presentation capacity.

An important feature of microbial infections is the ability of the microorganisms to interfere with and modulate the induction of host immune reactions. However, little is known about the effects of broad host range pathogens such as Listeria monocytogenes on similar cell types in different hosts. Here we examine the effects of the human and animal pathogen L. monocytogenes on human dendritic cells (DC) since this type of cells is essential for the initiation of immune responses. Listeria are phagocytosed efficiently by immature human DC and the bacteria escape from the phagolysosome quickly. Lack of the pore-forming activity of listeriolysin, which was found to be essential for the vacuolar escape of this bacterium in other cell types, retarded but did not prevent egress from the vacuole. Treatment of cultures of immature DC with L. monocytogenes resulted in rapid changes in morphology and cellular constitution followed by maturation of the DC. This could be judged by the appearance of maturation-specific cell surface markers. Antigen presentation to CD4 T cells was apparently not impaired by the infection. These results are in clear contrast to results obtained previously in the mouse system (Guzman et al., Mol. Microbiol. 1996. 20: 119 - 126; Darji et al., Eur. J. Immunol. 1997. 27: 1696 - 1703.).

Genome organization and the evolution of the virulence gene locus in Listeria species.

The chromosomal region of Listeria monocytogenes harboring the gene cluster prfA-plcA-hly-mpl-actA-plcB (virulence gene cluster; vgc) harbors virulence genes critical for the survival of the bacteria following infection. Previous studies have implicated it as an ancestral pathogenicity island, derivatives of which are present in the species L. ivanovii and L. seeligeri, but absent in non-pathogenic species such as L. innocua. We cloned the corresponding region from L. innocua and L. welshimeri and compared its sequences to those from L. monocytogenes, L. ivanovii and L. seeligeri. The analysis allowed exact determination of delineation and size of the vgc and suggests that these genes may have been acquired by bacteriophage transduction. Thus, here we present an alternative view of the evolution of Listeria spp. and suggest that L. monocytogenes may be the primordial species of this genus.

Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex.

The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.

[Rapid and reliable detection of multiresistent Staphylococcus aureus (MRSA) by multiplex PCR]. / Schneller und zuverlässiger Nachweis multiresistenter Staphylococcus aureus (MRSA) durch Multiplex-PCR.

BACKGROUND AND OBJECTIVE: Staphylococci are widespread pathogens and are frequently associated with nosocomial infections. Many hospitals struggle with increasing amounts of methicillin-resistant Staphylococcus aureus (MRSA) which are "multiresistant" against all betalactam antibiotics. Often, applicable antibiotics for treatment are only glycopeptides like vancomycin and teicoplanin. In addition, MRSA infected patients require expensive intensive isolation measures and strict hygiene. To efficiently prevent dissemination of these pathogens rapid and reliable identification and a close collaboration between clinicians and microbiologists are required. The purpose of our study was to set up a rapid and reliable identification procedure for MRSA by the amplification of specific gene determinants by PCR in order to to efficiently support therapy and eradication of the pathogen. METHODS: 153 strains of staphylococci isolated from in-patients of the hospital of the Justus-Liebig University of Giessen were examined. The femB gene was used to differentiate between Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS), a gene which allows the species-specific identification of methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA). Additionally, MRSA harbor the mecA gene encoding methicillin-resistance, which is absent in MSSA strains. RESULTS: Using a multiplex PCR with femB and mecA gene-specific oligonucleotides MRSA strains were unequivocally detected within 3 hours. The femB gene was detected in all 102 strains of S. aureus but in none of the 51 CNS. The mecA determinant was detected in 12 S. aureus. Among these, 11 strains were phenotypically methicillin-resistant and one strain was susceptible. The methicillin-resistance of this particular mecA-positive/methicillin-susceptible strain (cryptic MRSA) was inducible by cultivation on agar plates supplemented with flucloxacillin. CONCLUSIONS: The described method specifically detects S. aureus and identifies phenotypical and cryptic MRSA. These cryptic MRSA are of particular relevance since they are undetectable using common phenotypically based detection methods. It is conceivable that the methicillin resistance of these strains is induced under antibiotic therapy with flucloxacillin and that the mec-encoded feature of methicillin-resistance can be transferred to previously methicillin-susceptible strains. Using the reliable detection of these strains by PCR, failure of flucloxacillin therapy is avoidable.

Physical and genetic map of the Listeria monocytogenes EGD serotype 1/2a chromosome.

Listeria monocytogenes is a facultative intracellular pathogen responsible for both invasive and non-invasive food-borne illness in animals and humans. In this study, macrorestriction analysis following pulsed-field gel electrophoresis was used to show that Listeria monocytogenes serovar 1/2a strain EGD has a single chromosome containing eight NotI fragments of 1100, 850, 365, 320, 275, 40, 30 and 20 kb in size and 11 AscI fragments of 860, 470, 410, 360, 320, 250, 110, 80, 50, 30 and 20 kb. The total genome therefore comprises 3000 +/- 50 kb. The creation of a physical and genetic map of the Listeria genome was achieved by generating NotI linking clones and their use in subsequent hybridisation analysis. Using isogenic mutants harbouring additional artificial NotI restriction sites, we were able to precisely map the positions of all currently known virulence genes on the chromosome.

Phosphatidylcholine-specific phospholipase C from Listeria monocytogenes is an important virulence factor in murine cerebral listeriosis.

Meningoencephalitis is a serious and often fatal complication of Listeria monocytogenes infection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion of L. monocytogenes into cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread of L. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (DeltainlAB2)- and a PlcB (DeltaplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT) L. monocytogenes EGD. Listeria strains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384-2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the DeltaplcB2 strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the DeltainlAB2 strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with the DeltaplcB2 strain compared to that of mice infected with the WT or DeltainlAB2 strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the DeltaplcB2 mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of the Listeria mutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murine L. monocytogenes meningoencephalitis.

Two distinct phospholipases C of Listeria monocytogenes induce ceramide generation, nuclear factor-kappa B activation, and E-selectin expression in human endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD delta plcA and EGD delta plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD delta plcA or EGD delta plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-kappa B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-kappa B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.