[Recurrent intracerebral haemorrhage in a 24-year-old female patient]. / Rezidivierende intrazerebrale Blutungen bei einer 24-jährigen Patientin.

A 24-year-old female patient from Sierra Leone was referred to the authors' hospital after several unclear intracerebral bleeding events and an echogenic structure on the aortic valve. The patient was receiving oral anticoagulation therapy due to paroxysmal atrial fibrillation and left ventricular noncompaction. Fluorescence in situ hybridization in combination with polymerase chain reaction and sequencing revealed infective endocarditis of the mitral and aortic valve caused by Bartonella quintana. In retrospect, the intracerebral bleeding events could be identified as septic emboli with secondary haemorrhagic transformation under anticoagulation therapy. The patient showed significant clinical improvement and no further bleeding events occurred after receiving biological mitral and aortic valve replacement and several weeks of doxycycline and gentamicin antibiotic therapy.

Effect of daptomycin and vancomycin on Staphylococcus epidermidis biofilms: An in vitro assessment using fluorescence in situ hybridization.

Colonization of in-dwelling catheters by microbial biofilms is a major concern in patient health eventually leading to catheter-related blood stream infections. Biofilms are less susceptible to standard antibiotic therapies that are effective against planktonic bacteria. Standard procedure for the detection of microorganisms on the catheter tip is culture. However, viable but non-culturable cells (VBNCs) may be missed. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) as an indicator to visualize and quantify the effect of the antibiotics daptomycin and vancomycin on biofilms in situ. We established an in vitro catheter biofilm model of Staphylococcus epidermidis biofilms on polyurethane catheters. Biofilm activity was measured by FISH and correlated to colony forming units (CFU) data. Digital image analysis was used for quantification of total biofilm mass and the area of the FISH positive biofilm cells. FISH showed a pronounced effect of both antibiotics on the biofilms, with daptomycin having a significantly stronger effect in terms of both reduction of biofilm mass and number of FISH-positive cells. This supports the anti-biofilm capacity of daptomycin. Interestingly, neither antibiotic was able to eradicate all of the FISH-positive cells. In summary, FISH succeeded in visualization, quantification, and localization of antibiotic activity on biofilms. This technique adds a new tool to the arsenal of test systems for anti-biofilm compounds. FISH is a valuable complementary technique to CFU since it can be highly standardized and provides information on biofilm architecture and quantity and localization of survivor cells.

[The significance of biofilm for the treatment of infections in orthopedic surgery : 2017 Update]. / Bedeutung des Biofilms für die Infektbehandlung in der Unfallchirurgie : Update 2017.

BACKGROUND: The increase in endoprosthetic and osteosynthetic surgical treatment is associated with a simultaneous increase in implant-associated infections (surgical site infections, SSI). Biofilms appear to play a significant role in the diagnosis and treatment of these infections and heavily contaminated wounds. This article aims to provide a current overview of biofilm and its relevance in orthopedic surgery. MATERIALS AND METHODS: A computer-assisted literature search of MedLine (PubMed) was performed using key word combinations with "biofilm" (as of March 2017). RESULTS: Biofilm, a polymicrobial organization and life form surrounded by a polysaccharide matrix, refers to an adaptation strategy of bacteria in unfavorable living conditions (e. g. under antibiotic therapy). Biofilms can develop after 6 h in highly contaminated wounds. In acute and chronic infections, biofilms can occur in 30-80 % of the cases. Only planktonic bacteria (high metabolic activity, cultivable) can be detected in standard microbiological cultures, biofilms, however, cannot. Molecular microscopic methods, such as fluorescence in situ hybridization (FISH), enable the detection of bacteria in biofilms. The core concepts of anti-biofilm therapy include the prevention of biofilm and early surgical debridement, followed by the local and/or systemic administration of antibiotics as well as the local application of antiseptics. CONCLUSIONS: The development of biofilm should be anticipated in strongly contaminated wounds as well as in acute and chronic infection sites. The best strategy to combat biofilms is to prevent their development. Standard microbiological culture methods do not enable the detection of biofilm. Therefore, the implementation of molecular biological detection methods (z. B. FISH) is important. Further anti-biofilm strategies are being investigated experimentally, but there are no real options for clinical use as of yet.

Is Ureaplasma spp. the leading causative agent of acute chorioamnionitis in women with preterm birth?

OBJECTIVES: Aim of this study was to detect microorganisms in fetal membranes and placental tissue in preterm chorioamnionitis by combining fluorescence in situ hybridization (FISH) with broad range PCR. The combination of the two molecular techniques enables identification and localization of the microorganisms within the tissue, confirming their clinical relevance. METHODS: In a prospective cohort study, we compared 31 women with preterm premature rupture of membranes or preterm labour and preterm delivery by caesarean section with a control group of 26 women undergoing elective caesarean section at term. Fetal membranes and placental tissue were analysed by FISH and broad range 16S rRNA-gene PCR and sequencing. RESULTS: For 20 women in the preterm group, caesarean section was performed because of a clinical diagnosis of chorioamnionitis. Microorganisms were detected in the tissues by both molecular techniques in 11 out of 20 women. Among those, Ureaplasma spp. was most abundant, with five cases that remained culture-negative and would have been missed by routine diagnostic procedures. Other infections were caused by Staphylococcus aureus, Streptococcus mitis or Escherichia coli. FISH and PCR were negative for all women without suspected chorioamnionitis and for the control group. CONCLUSIONS: Combination of FISH with broad-range PCR and sequencing permitted unambiguous identification of the causative microorganisms in chorioamnionitis. The high prevalence of Ureaplasma spp. should lead to a re-evaluation of its clinical significance and possible therapeutic consequences.

Moraxella catarrhalis induces CEACAM3-Syk-CARD9-dependent activation of human granulocytes.

The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria-induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-3 is linked to NF-κB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by enzyme-linked immunosorbent assay for chemokines. NF-κB activation was determined using a luciferase reporter gene assay and chromatin-immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by M. catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro-inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF-κB pathway via Syk and the CARD9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM-like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis.

Fluorescence in situ hybridization for identification of microorganisms in acute chorioamnionitis.

The relevance of microorganisms in preterm birth is still under discussion. Using a diagnostic fluorescence in situ hybridization probe panel, we visualized Staphylococcus aureus and Streptococcus mitis group in two cases of acute chorioamnionitis. This technique provides spatial resolution and quantity of bacteria, clarifying the epidemiology and pathogenic pathways of acute chorioamnionitis.

Fluorescence in situ hybridization to improve the diagnosis of endocarditis: a pilot study.

Infective endocarditis is a rare but life-threatening disease associated with high mortality. Early diagnosis of the causative microorganism is critical to patient outcome. However, conventional diagnostic methods are often unsatisfactory in achieving this goal. As a proof of concept, we applied fluorescence in situ hybridization (FISH) for detection and identification of bacteria in histological sections of heart valves. Biopsy specimens from 54 suspected endocarditis patients were obtained during valve surgery and analysed via FISH. Specimens were screened with a probe panel that identifies the most common bacteria implicated in endocarditis. Results were compared with those of culture-based diagnostics and clinical data. Discrepant results were subjected to comparative sequence analysis of PCR-amplified 16S rRNA genes. FISH detected bacteria in 26 of the 54 heart valves. FISH allowed successful diagnosis of infective endocarditis in five of 13 blood culture-negative cases and in 11 of 37 valve culture-negative cases, showing the bacteria within their histological context. This technique allows the simultaneous detection and identification of microorganisms at the species or genus level directly from heart valves and might be a valuable tool for diagnosis of endocarditis.

[Melioidosis-like disease: infection with Burkholderia cocovenenans as rare differential diagnosis for Lymphadenitis colli]. / Melioidosis-like disease: Infektion mit Burkholderia cocovenenans als seltene Differenzialdiagnose bei Lymphadenitis colli.

INTRODUCTION: Neoplasms, autimmune disorders and infectious diseases as differential diagnoses of cervical lymphadenopathies also require the consideration of rare causes. CASE REPORT: A 25-year-old patient presented for further diagnosis and treatment of a colliquating, high febrile cervical lymphadenopathy. The patient from Thailand who had been living in Germany for 8 years reported she worked as rice farmer during the 1980s. Examination showed a vast physical condition with severe weight loss, joint- and swallowing aches which did not respond to high doses of parenteral antibiotic treatment. The histology of a lymph node revealed a necrotizing lymphadenitis, lymphoma were excluded. During further complications (sepsis, splenic and intracerebral abscesses and osteomyelitis) multiple different cytologic samples from lympoid tissue, different wound lesions and bronchial secretion microscopically showed non-fermenting, gram-negative rods by 16S-rDNA-analysis identified as Burkholderia cocovenenans/gladioli. Thus a melioidosis-like disease (endemic in south east asia) was diagnosed. Responsible for the severe course with a lethal recurrence despite antibiotic treatment was the patients additional immune defect (anti-interferone-gamma-autoantibodies). SUMMARY: Travelling history informations become more and more important considering increasing long-distance travelling and worldwide migration movements. Unclear inflammatory/infectious diseases require early interdisciplinary treatment. Detailed informations for the pathologist facilitate the diagnosis.

Chronic periodontal disease is associated with single-nucleotide polymorphisms of the human TLR-4 gene.

Periodontitis is an inflammatory disease affecting the connective tissue surrounding the teeth leading to tooth loss. Pathogens associated with periodontitis interact with Toll-like receptors (TLRs) to induce cytokines causing and aggravating disease. We screened 197 individuals suffering from generalized periodontitis for the presence of Asp299Gly and Thr399Ile of TLR-4 as well as Arg753Gln of TLR-2 in comparison to matched controls. Single-nucleotide polymorphisms (SNPs) of TLR-4 were elevated among patients (odd's ratio 3.650, 95% CI 1.573-8.467, P < or = 0.0001), while no difference was observed for TLR-2. TLR-4 SNPs were correlated with chronic periodontitis (odd's ratio 5.562, 95% CI 2.199-14.04, P < or = 0.0001), but not with aggressive periodontitis. This observation was confirmed employing a group of periodontally healthy probands over 60 years of age. These data demonstrate that genetic variants of TLR-4 may act as risk factors for the development of generalized chronic periodontitis in humans.

Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare test.

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.

Treponema putidum sp. nov., a medium-sized proteolytic spirochaete isolated from lesions of human periodontitis and acute necrotizing ulcerative gingivitis.

So far, little phenotypic heterogeneity has been detected in cultured oral treponemes with trypsin-like proteolytic activity, and all have been assigned to the species Treponema denticola. However, comparisons of protein patterns and antigen expression in our collection of proteolytic oral treponemes occasionally identified isolates with a unique phenotype; e.g. strain OMZ 830 (=ATCC 700768), which qualified as a 'pathogen-related oral spirochaete' due to the presence of a approximately 37 kDa protein reactive with the Treponema pallidum FlaA-specific mAb H9-2. In addition to such single isolates, a homogeneous group of seven independent strains is described that were highly motile, medium-sized, proteolytic but asaccharolytic spirochaetes and were cultured from human gingivitis, periodontitis and acute necrotizing ulcerative gingivitis in medium OMIZ-Pat supplemented with 1% human serum and antibiotics. Growth of these spirochaetes in OMIZ-Pat was not dependent on, but was stimulated by, human or bovine serum. Carbohydrates were neither required nor stimulatory for growth. The protein and antigen patterns of total cell extracts of these organisms separated by SDS-PAGE were distinct from those of all previously cultured spirochaetes, with highest similarity to T. denticola. The novel spirochaete has a 2 : 4 : 2 arrangement of the periplasmic flagella, similar to T. denticola. However, the flagellin pattern as detected by immunostaining or glycan staining of Western blots readily distinguished the novel group from T. denticola. Also, distinct from reference strains of T. denticola, none of the novel isolates displayed sialidase or dentilisin activities, both of which are expressed by most strains of T. denticola. Trypsin-like activity and other enzymes as detected by API ZYM test were similar to those of T. denticola. The status of a novel species is supported by the 16S rRNA gene sequence, with 98.5% similarity to its closest cultured relative, T. denticola. The name Treponema putidum sp. nov. is proposed (type strain OMZ 758T=ATCC 700334T=CIP 108088T).

Chemotaxis in pathogenic spirochetes: directed movement toward targeting tissues?

Chemotaxis is an important feature of motile organisms that allows navigation through various environments. It enables them to detect nutrients and to avoid unfavorable or dangerous conditions. Motility and chemotaxis are widely acknowledged as important virulence factors for pathogenic bacteria. In this review, we try to explore the role of chemotaxis in the pathogenesis of spirochetes. Chemotaxis might be involved in tissue identification and penetration, and represents a possible mechanism for evasion of the host's immune defense. The recent development of genetic tools for pathogenic spirochetes and "tracking" techniques, employing fluorescent in situ hybridization (FISH), could revolutionize our understanding of the importance of chemotaxis for infection and persistence of these bacteria in their host.

Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms.

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.

A novel technique for monitoring the development of bacterial biofilms in human periodontal pockets.

A new technique is presented for analyzing subgingival bacterial plaque. Different materials (polytetrafluoroethylene, gold, dentin) kept for several days in periodontal pockets of patients suffering from periodontitis were analyzed by electron microscopy and fluorescence in situ hybridization (FISH). Those parts of the carriers extending into the deepest zone of the pockets were predominantly colonized by spirochetes and Gram-negative bacteria whereas those segments in contact with a shallower region were colonized by streptococci. Independent of the material used, the bacterial colonization of the carriers appears to be similar. FISH using eubacteria- and species-specific oligonucleotides on semi-thin cross-sections of the carriers in combination with confocal laser scanning microscopy allowed detailed analysis of the architecture of biofilms and identification of putative periodontal pathogens with single cell resolution.

Phylogenetic analysis of rhizosphere-associated beta-subclass proteobacterial ammonia oxidizers in a municipal wastewater treatment plant based on rhizoremediation technology.

In wastewater treatment plants based on the rhizosphere zone (rhizoremediation technology), ammonia-oxidizing bacteria (AOB) play an important role in the removal of fixed nitrogen. However, the diversity of these bacteria in rhizoremediation wastewater treatment plants is largely unknown. We employed direct PCR amplification and cloning of 16S rRNA genes to determine the phylogenetic affiliation of AOB occurring in root and soil samples of a wastewater treatment plant (Merzdorf plant, Brandenburg, Germany). 16S rDNA clone libraries were screened by hybridization using an oligonucleotide probe specific for AOB of the beta subclass of proteobacteria. Comparative sequence analysis of all hybridization-positive clones revealed that the majority of rDNA sequences was affiliated to members of the genus Nitrosospira and formed a novel subcluster (SM cluster), whereas only three sequences were most closely related to Nitrosomonas species. Affiliation of the novel Nitrosospira-like sequences with those of isolates from soil and rhizosphere suggests that phylogenetic clusters reflect physiological differences between members of this genus.

A case of septicemia with Pediococcus acidilactici after long-term antibiotic treatment.

Presented here is a case of septicemia caused by an uncommon, multiresistant, gram-positive microorganism (Pediococcus acidilactici) after long-term antibiotic treatment. Pediococcus spp. are rarely cultivated from clinical specimens, and species differentiation is difficult due to the paucity of phenotypic traits. In this case, a polyphasic approach consisting of phenotypic and molecular genetic analyses was used, and the identification of Pediococcus acidilactici was conclusive. Precise identification and antimicrobial susceptibility testing of rarely isolated bacteria are required in order to provide adequate treatment to infected patients and to determine the pathogenic role of these organisms.

Treponema lecithinolyticum sp. nov., a small saccharolytic spirochaete with phospholipase A and C activities associated with periodontal diseases.

Strong phospholipase A (PLA) and phospholipase C (PLC) activities as potential virulence factors are the outstanding characteristics of eight strains of small oral spirochaetes isolated from deep periodontal lesions. By qualitative dot-blot DNA-DNA hybridization and 16S rDNA sequence comparison, these spirochaetes form a distinct phylogenetic group, with Treponema maltophilum as its closest cultivable relative. Growth of these treponemes, cells of which contain two endoflagella, one at each pole, was autoinhibited by the PLA-mediated production of lysolecithin unless medium OMIZ-Pat was prepared without lecithin. N-Acetylglucosamine was essential and D-ribose was stimulatory for growth. All isolates were growth-inhibited when 1% foetal calf serum was added to the medium. Growth on agar plates supplemented with human erythrocytes produced haemolysis. In addition to PLA and PLC, the new isolates displayed strong activities of alkaline and acid phosphatases, beta-galactosidase, beta-glucuronidase, N-acetyl-beta-glucosaminidase and sialidase, intermediate activities of C4- and C8-esterases, naphthol phosphohydrolase and alpha-fucosidase and a distinctive 30 kDa antigen detectable on Western blots. This phenotypically and genotypically homogeneous group is proposed as a novel species, Treponema lecithinolyticum sp. nov., with isolate OMZ 684T designated as the type strain. A molecular epidemiological analysis using a T. lecithinolyticum-specific probe showed this organism to be associated with affected sites when compared with unaffected sites of periodontitis patients. This association was more pronounced in patients with rapidly progressive periodontitis than in those with adult periodontitis.

Treponema brennaborense sp. nov., a novel spirochaete isolated from a dairy cow suffering from digital dermatitis.

A novel Treponema species was isolated from an ulcerative lesion of a cow suffering from digital dermatitis (DD), a disease which causes painful ulcerations along the coronary band. Among other anaerobic bacteria, high numbers of spirochaetes have been regularly found in DD lesions. Here data are presented of a spirochaete isolated from a DD ulcer. By chemotaxonomy, protein analysis and comparative 16S rDNA sequence analysis this isolate was classified as a treponeme that differed from all Treponema species described previously. The only isolate, DD5/3T, for which the name Treponema brennaborense is proposed, is designated the type strain of the novel species. The strain is a small, highly motile spirochaete that has two periplasmic flagella, one flagellum being attached at each cell pole. Strain DD5/3T exhibits alpha-glucosidase and N-acetyl-beta-glucosaminidase activity and growth is inhibited by rabbit serum. T. brennaborense was phylogenetically most closely related (89.5% 16S rRNA similarity) to Treponema maltophilum, an oral spirochaete isolated from a periodontitis patient.

Cloning and characterization of a gene (mspA) encoding the major sheath protein of Treponema maltophilum ATCC 51939(T).

The major sheath protein-encoding gene (mspA) of the oral spirochete Treponema maltophilum ATCC 51939(T) was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The mspA gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an mspA-specific probe showed no cross-reactivity with the msp gene from Treponema denticola.