Publications by authors named "Annette Moter"

74 Publications

Identification of anti-microbial peptides and traces of microbial DNA in infrainfundibular compartments of human scalp terminal hair follicles.

Eur J Dermatol 2021 Feb;31(1):22-31

Department of Dermatology and Allergy, Clinical Research Center for Hair and Skin Science, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Background: The upper follicular compartment, a well-known reservoir of cutaneous microbiota, constitutes a space for intensive cross-barrier dialogue. The lower follicle comprises the bulb and bulge, structures with relative immune-privileged status, crucial for physiological cycling, and widely considered to be microbial-free.

Objectives: Following our initial immunohistochemical screening for regulatory cytokines and defensin expression in anagen hair follicles, we aimed to confirm our results with a follow-up ELISA investigation. We postulated that exposure to microbial components may trigger expression, and thus opted to investigate microbial presence in this area.

Materials & Methods: We performed immunohistochemical staining for selected cytokines and antimicrobial peptides, and Gram and Giemsa staining on tissue sections from healthy individuals. Based on ELISA analyses, we confirmed a marked presence of IL-17A- and HBD2 in infrainfundibular compartments from plucked anagen hair follicles of 12 individuals (six females, six males; frontal and occipital scalp sites). 16S rRNA sequencing on microbial DNA extracted from lower follicles, as well as fluorescence in situ hybridization (FISH) were applied to explore bacterial presence in the infrainfundibular compartments.

Results: 16S rRNA sequencing yielded reproducible data of bacterial presence in infrainfundibular compartments of plucked scalp follicles; Lawsonella clevelandensis, Staphylococcaceae and Propionibacteriaceae were the most abundant bacteria. Also, FISH revealed biofilm structures formed by Cutibacterium acnes (formerly Propionibacterium acnes) and Staphylococcus sp. below the infundibulum.

Conclusion: As the skin microbiome largely influences the local immune system, the presence of bacteria in proximity to follicular immune-privileged areas may be of relevance to hair cycling in health and disease.
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http://dx.doi.org/10.1684/ejd.2020.3948DOI Listing
February 2021

Rothia aeria and Rothia dentocariosa as biofilm builders in infective endocarditis.

Int J Med Microbiol 2021 Feb 6;311(2):151478. Epub 2021 Feb 6.

Biofilmcenter, Institute for Microbiology, Infectious Diseases and Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany; MoKi Analytics GmbH, Berlin, Germany.

Background: Rothia sp. are Gram-positive bacteria in the class of Actinobacteria that are part of the physiological oral flora. In rare cases, Rothia aeria and Rothia dentocariosa can cause infective endocarditis (IE). The biofilm potential of Rothia in endocarditis is unknown.

Methods: Specimen from two cases of Rothia endocarditis were obtained during cardiac surgery. One of the patients suffered mitral valve IE from Rothia aeria. In the other case, IE of a prosthetic pulmonary valve was caused by Rothia dentocariosa. Fluorescence in situ hybridization (FISH) was used for visualization of microorganisms within heart valve tissues in combination with PCR and sequencing (FISHseq).

Results: The two heart valve specimens featured mature biofilms of bacteria that were identified by FISHseq as Rothia aeria and Rothia dentocariosa, respectively. FISH showed in situ biofilms of both microorganisms that feature distinct phenotypes for the first time ex vivo. Both of our reported cases were treated successfully by heart valve surgery and antibiotic therapy using beta-lactam antibiotics.

Conclusion: The biofilm potential of Rothia sp. must be taken into account. The awareness of Rothia aeria and Rothia dentocariosa as rare but relevant pathogens for infective endocarditis must be raised. Use of biofilm-effective antibiotics in Rothia IE should be discussed.
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http://dx.doi.org/10.1016/j.ijmm.2021.151478DOI Listing
February 2021

Quality Control in Diagnostic Fluorescence In Situ Hybridization (FISH) in Microbiology.

Methods Mol Biol 2021 ;2246:301-316

Biofilmcenter, Institute for Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

This overview addresses fluorescence in situ hybridization (FISH) in a diagnostic microbiology setting with its associated problems and pitfalls and how to control them, but also the advantages and opportunities the method offers. This article focuses mainly on diagnostic FISH assays on tissue sections and on techniques and experiences in our laboratory. FISH in a routine diagnostic setting in microbiology requires strict quality control measures to ensure consistent high-quality and reliable assay results. Here, for the first time, we define quality control requirements for microbiological diagnostic FISH applications and discuss their impact and possible future developments of the FISH technique for infection diagnostics. We focus on diagnosis of biofilm-associated infections including infective endocarditis, oral biofilms, and device-associated infections as well as infections due to fastidious or yet uncultured microorganisms like Treponema spp., Tropheryma whipplei, Bartonella, Coxiella burnetii, or Brachyspira.
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http://dx.doi.org/10.1007/978-1-0716-1115-9_20DOI Listing
March 2021

Cutibacterium avidum resists surgical skin antisepsis in the groin-a potential risk factor for periprosthetic joint infection: a quality control study.

Antimicrob Resist Infect Control 2021 02 1;10(1):27. Epub 2021 Feb 1.

Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.

Background: The skin commensal Cutibacterium avidum has been recognized as an emerging pathogen for periprosthetic joint infections (PJI). One currently assumes that the early occurring PJIs are a consequence of skin commensals contaminating the peri-implant tissue during surgery. We addressed whether standard skin antisepsis with povidone-iodine/alcohol before total hip arthroplasty (THA) is effective to eliminate colonizing bacteria with focus on C. avidum.

Methods: In a single-center, prospective study, we screened all patients for skin colonizing C. avidum in the groin before THA. Only in the patients positive for C. avidum, we preoperatively repeated skin swabs after the first and third skin antisepsis and antibiotic prophylaxis. We also obtained dermis biopsies for microbiology and fluorescence in situ hybridization (FISH).

Results: Fifty-one out of 60 patients (85%) were colonized on the skin with various bacteria, in particular with C. avidum in 12 out of 60. Skin antisepsis eliminated C. avidum in eight of ten (20%) colonized patients undergoing THA. Deeper skin (dermis) biopsies were all culture negative, but FISH detected single positive ribosome-rich C. avidum in one case near sweat glands.

Conclusion: Standard skin antisepsis was not effective to completely eliminate colonizing C. avidum on the skin in the groin of patients undergoing THA. Colonizing with C. avidum might pose an increased risk for PJI when considering a THA. Novel more effective antisepsis strategies are needed. Trial registration No clinical trial.
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http://dx.doi.org/10.1186/s13756-021-00883-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852298PMC
February 2021

New model in diabetic mice to evaluate the effects of insulin therapy on biofilm development in wounds.

GMS Interdiscip Plast Reconstr Surg DGPW 2020 23;9:Doc06. Epub 2020 Dec 23.

Department of Orthopedics, Trauma and Plastic Surgery, University Hospital Leipzig, Germany.

Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7 +/+ Lepr/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with ATCC 49230 (10 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistochemical staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19). The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. We developed a novel model to assess interactions between blood glucose level and -induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.
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http://dx.doi.org/10.3205/iprs000150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818390PMC
December 2020

Fluorescence In Situ Hybridization and Polymerase Chain Reaction to Detect Infections in Patients With Left Ventricular Assist Devices.

ASAIO J 2021 May;67(5):536-545

Biofilmcenter, Institute of Microbiology, Infectious Diseases and Immunology, Charité - University Medical Center Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.

The development of driveline infections following left ventricular assist device (LVAD) implantation remains a major problem. We investigated the impact of fluorescence in situ hybridization (FISH) combined with 16S rRNA gene sequencing on the diagnosis of driveline infections. LVAD drivelines (n = 61) from 60 consecutive patients were obtained during LVAD explantation and subjected to FISH analysis. 16S rRNA gene polymerase chain reaction (PCR) and sequencing to identify the microorganisms were performed. Results were compared with those of a standard microbiological culture. The reasons for pump removal were heart transplantation (n = 22), weaning (n = 14), pump exchange due to pump thrombosis (n = 12), technical problems (n = 7), or death (n = 5). Of the 60 patients, 26 exhibited clinical signs of a VAD-specific infection, while 34 (with 35 drivelines) showed no clinical signs of infection before explantation. The spectrum of identified pathogens differed between FISH/PCR and conventional microbiological diagnostics. In general, the bacterial spectrum was more diverse in FISH/PCR as compared with conventional microbiology, which more often showed only typical skin flora (coagulase-negative staphylococci and Corynebacteriaceae). In addition to identifying the species, FISH/PCR provided information about the spatial distribution and invasiveness of the microorganisms. Cultures usually represent the only source of microbiological information for clinicians and often prove to be unsatisfactory in complex LVAD cases. FISH/PCR not only identified a greater number and variety of microorganisms than standard culture did, but it also provided information about the number, localization, and biofilm state of the pathogens, making it a useful tool for diagnosing the specific cause of LVAD driveline infections.
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http://dx.doi.org/10.1097/MAT.0000000000001260DOI Listing
May 2021

Spread of Multidrug-Resistant Bacteria by Moth Flies from Hospital Waste Water System.

Emerg Infect Dis 2020 08;26(8):1893-1898

We documented and analyzed moth fly occurrence and spread of multidrug-resistant bacteria in a tertiary care hospital in Germany. The moth flies (Clogmia albipunctata) bred in the sewage system, then moved into the hospital, carrying biofilm and multidrug-resistant bacteria on their feet. Subsequently, the hospital developed a pest control protocol.
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http://dx.doi.org/10.3201/eid2608.190750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392454PMC
August 2020

Bacterial biofilms in infective endocarditis: an in vitro model to investigate emerging technologies of antimicrobial cardiovascular device coatings.

Clin Res Cardiol 2021 Mar 22;110(3):323-331. Epub 2020 May 22.

Biofilmcenter, Department of Microbiology, Infectious Diseases and Immunology, Charité, Universitaetsmedizin Berlin, Berlin, Germany.

Objective: In spite of the progress in antimicrobial and surgical therapy, infective endocarditis (IE) is still associated with a high morbidity and mortality. IE is characterized by bacterial biofilms of the endocardium, especially of the aortic and mitral valve leading to their destruction. About one quarter of patients with formal surgery indication cannot undergo surgery. This group of patients needs further options of therapy, but due to a lack of models for IE prospects of research are low. Therefore, the purpose of this project was to establish an in vitro model of infective endocarditis to allow growth of bacterial biofilms on porcine aortic valves, serving as baseline for further research.

Methods And Results: A pulsatile two-chamber circulation model was constructed that kept native porcine aortic valves under sterile, physiologic hemodynamic and temperature conditions. To create biofilms on porcine aortic valves the system was inoculated with Staphylococcus epidermidis PIA 8400. Aortic roots were incubated in the model for increasing periods of time (24 h and 40 h) and bacterial titration (1.5 × 10 CFU/mL and 1.5 × 10 CFU/mL) with 5 L cardiac output per minute. After incubation, tissue sections were analysed by fluorescence in situ hybridization (FISH) for direct visualization of the biofilms. Pilot tests for biofilm growth showed monospecies colonization consisting of cocci with time- and inocula-dependent increase after 24 h and 40 h (n = 4). In n = 3 experiments for 24 h, with the same inocula, FISH visualized biofilms with ribosome-containing, and thus metabolic active cocci, tissue infiltration and similar colonization pattern as observed by the FISH in human IE heart valves infected by S. epidermidis.

Conclusion: These results demonstrate the establishment of a novel in vitro model for bacterial biofilm growth on porcine aortic roots mimicking IE. The model will allow to identify predilection sites of valves for bacterial adhesion and biofilm growth and it may serve as baseline for further research on IE therapy and prevention, e.g. the development of antimicrobial transcatheter approaches to IE.
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http://dx.doi.org/10.1007/s00392-020-01669-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907033PMC
March 2021

Aerococcus urinae - A potent biofilm builder in endocarditis.

PLoS One 2020 23;15(4):e0231827. Epub 2020 Apr 23.

Biofilmzentrum, Dept. of Microbiology, Infectious Disease and Immunology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

The diagnosis of infective endocarditis (IE) remains a challenge. One of the rare bacterial species recently associated with biofilms and negative cultures in infective endocarditis is Aerococcus urinae. Whether the low number of reported cases might be due to lack of awareness and misidentification, mainly as streptococci, is currently being discussed. To verify the relevance and biofilm potential of Aerococcus in endocarditis, we used fluorescence in situ hybridization to visualize the microorganisms within the heart valve tissue. We designed and optimized a specific FISH probe (AURI) for in situ visualization and identification of A. urinae in sections of heart valves from two IE patients whose 16S rRNA gene sequencing had deteced A. urinae. Both patients had a history of urinary tract infections. FISH visualized impressive in vivo grown biofilms in IE, thus confirming the potential of A. urinae as a biofilm pathogen. In both cases, FISH/PCR was the only method to unequivocally identify A. urinae as the only causative pathogen for IE. The specific FISH assay for A. urinae is now available for further application in research and diagnostics. A. urinae should be considered in endocarditis patients with a history of urinary tract infections. These findings support the biofilm potential of A. urinae as a virulence factor and are meant to raise the awareness of this pathogen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231827PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180067PMC
July 2020

Fluorescence in situ hybridization for identification and visualization of microorganisms in infected heart valve tissue as addition to standard diagnostic tests improves diagnosis of endocarditis.

Interact Cardiovasc Thorac Surg 2019 11;29(5):678-684

Department of Cardiac Surgery, Hospital Bogenhausen, Munich, Germany.

Objectives: In infective endocarditis (IE), identification of the causative organism and consecutive treatment are crucial for patient survival. Although the macroscopic aspect resembles infected tissue, standard diagnostic tests often fail to allow one to identify bacteria. Fluorescence in situ hybridization (FISH) is a molecular, culture-independent technique that allows one to identify and visualize microorganisms within tissue and to recognize their morphology, number and activity. We analysed the diagnostic benefit of FISH/polymerase chain reaction (PCR) by comparing its results to those of standard diagnostic tests.

Methods: From September 2015 to April 2018, 128 patients underwent first-time or redo valve surgery to treat IE. Patients were designated according to the modified Duke criteria as definite (n = 61), possible (n = 34) or rejected (n = 33) IE. Tissue specimens obtained intraoperatively were analysed using FISH/PCR in addition to undergoing standard diagnostic testing and PCR alone.

Results: We used blood cultures to detect microorganisms in 67/128 patients; valve cultures, in 34/128; PCR, in 67/128; histopathological diagnosis showed IE in 72/128 cases. We were able to detect microorganisms in 103/128 cases using FISH/PCR, with 55/61 in definite IE. Furthermore, we were able to identify 26 cases of bacterial biofilm using FISH/PCR, despite antibiotic treatment of 61 in the definite, 13 in the possible and 1 in the rejected group, including 8/33 patients in the rejected group with active bacteria. In all cases, the patient's therapy was altered.

Conclusions: FISH/PCR was used to identify microorganisms in cases in which standard diagnostic tests failed to provide sufficient results for various reasons. Furthermore, FISH/PCR enabled us to identify bacterial biofilms and to differentiate between active versus degraded bacteria, thus indicating the impact of treatment. Therefore, we suggest FISH/PCR as an additional diagnostic tool in IE alongside standard diagnostic tests.
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http://dx.doi.org/10.1093/icvts/ivz159DOI Listing
November 2019

Reply to Tison and Saraux.

Clin Infect Dis 2019 08;69(5):905

Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Germany.

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http://dx.doi.org/10.1093/cid/ciz095DOI Listing
August 2019

Evaluating Efficacy of Antimicrobial and Antifouling Materials for Urinary Tract Medical Devices: Challenges and Recommendations.

Macromol Biosci 2019 05 18;19(5):e1800384. Epub 2019 Mar 18.

University of Groningen, University Medical Center Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

In Europe, the mean incidence of urinary tract infections in intensive care units is 1.1 per 1000 patient-days. Of these cases, catheter-associated urinary tract infections (CAUTI) account for 98%. In total, CAUTI in hospitals is estimated to give additional health-care costs of £1-2.5 billion in the United Kingdom alone. This is in sharp contrast to the low cost of urinary catheters and emphasizes the need for innovative products that reduce the incidence rate of CAUTI. Ureteral stents and other urinary-tract devices suffer similar problems. Antimicrobial strategies are being developed, however, the evaluation of their efficacy is very challenging. This review aims to provide considerations and recommendations covering all relevant aspects of antimicrobial material testing, including surface characterization, biocompatibility, cytotoxicity, in vitro and in vivo tests, microbial strain selection, and hydrodynamic conditions, all in the perspective of complying to the complex pathology of device-associated urinary tract infection. The recommendations should be on the basis of standard assays to be developed which would enable comparisons of results obtained in different research labs both in industry and in academia, as well as provide industry and academia with tools to assess the antimicrobial properties for urinary tract devices in a reliable way.
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http://dx.doi.org/10.1002/mabi.201800384DOI Listing
May 2019

Recommendations for design and conduct of preclinical in vivo studies of orthopedic device-related infection.

J Orthop Res 2019 02 21;37(2):271-287. Epub 2019 Feb 21.

AO Research Institute Davos, Clavadelerstrasse 8, 7270, Davos Platz, Switzerland.

Orthopedic device-related infection (ODRI), including both fracture-related infection (FRI) and periprosthetic joint infection (PJI), remain among the most challenging complications in orthopedic and musculoskeletal trauma surgery. ODRI has been convincingly shown to delay healing, worsen functional outcome and incur significant socio-economic costs. To address this clinical problem, ever more sophisticated technologies targeting the prevention and/or treatment of ODRI are being developed and tested in vitro and in vivo. Among the most commonly described innovations are antimicrobial-coated orthopedic devices, antimicrobial-loaded bone cements and void fillers, and dual osteo-inductive/antimicrobial biomaterials. Unfortunately, translation of these technologies to the clinic has been limited, at least partially due to the challenging and still evolving regulatory environment for antimicrobial drug-device combination products, and a lack of clarity in the burden of proof required in preclinical studies. Preclinical in vivo testing (i.e. animal studies) represents a critical phase of the multidisciplinary effort to design, produce and reliably test both safety and efficacy of any new antimicrobial device. Nonetheless, current in vivo testing protocols, procedures, models, and assessments are highly disparate, irregularly conducted and reported, and without standardization and validation. The purpose of the present opinion piece is to discuss best practices in preclinical in vivo testing of antimicrobial interventions targeting ODRI. By sharing these experience-driven views, we aim to aid others in conducting such studies both for fundamental biomedical research, but also for regulatory and clinical evaluation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:271-287, 2019.
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http://dx.doi.org/10.1002/jor.24230DOI Listing
February 2019

Potential Role for Urine Polymerase Chain Reaction in the Diagnosis of Whipple's Disease.

Clin Infect Dis 2019 03;68(7):1089-1097

Institute of Pathology, Nephropathology Section, University Hospital Hamburg-Eppendorf.

Background: Whipple's disease (WD) is a rare infection with Tropheryma whipplei that is fatal if untreated. Diagnosis is challenging and currently based on invasive sampling. In a case of WD diagnosed from a kidney biopsy, we observed morphologically-intact bacteria within the glomerular capsular space and tubular lumens. This raised the questions of whether renal filtration of bacteria is common in WD and whether polymerase chain reaction (PCR) testing of urine might serve as a diagnostic test for WD.

Methods: We prospectively investigated urine samples of 12 newly-diagnosed and 31 treated WD patients by PCR. As controls, we investigated samples from 110 healthy volunteers and patients with excluded WD or acute gastroenteritis.

Results: Out of 12 urine samples from independent, therapy-naive WD patients, 9 were positive for T. whipplei PCR. In 3 patients, fluorescence in situ hybridization visualized T. whipplei in urine. All control samples were negative, including those of 11 healthy carriers with T. whipplei-positive stool samples. In our study, the detection of T. whipplei in the urine of untreated patients correlated in all cases with WD.

Conclusions: T. whipplei is detectable by PCR in the urine of the majority of therapy-naive WD patients. With a low prevalence but far-reaching consequences upon diagnosis, invasive sampling for WD is mandatory and must be based on a strong suspicion. Urine testing could prevent patients from being undiagnosed for years. Urine may serve as a novel, easy-to-obtain specimen for guiding the initial diagnosis of WD, in particular in patients with extra-intestinal WD.
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http://dx.doi.org/10.1093/cid/ciy664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424077PMC
March 2019

Detection of Tropheryma whipplei in stool samples by one commercial and two in-house real-time PCR assays.

Trop Med Int Health 2019 01 8;24(1):101-108. Epub 2018 Nov 8.

Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Rostock, Germany.

Objective: Tropheryma whipplei, the causative agent of Whipple's disease, can also be identified in stool samples of humans without systemic disease. It is much more frequently detected in human stool samples in tropical environments than in industrialized countries. PCR-screening has been applied for point prevalence studies and environmental assessments in tropical settings, but results depend on the applied assay. We compared one commercial qPCR kit with two well-described in-house assays for detection of T. whipplei from stool.

Methods: Residual materials from nucleic acid extractions of stool samples from two groups with presumably different prevalences and increased likelihood of being colonized or infected by T. whipplei were tested. One group comprised 300 samples from study participants from western Africa (group 1); the second group was of 300 returnees from tropical deployments (group 2). Each sample was assessed with all three qPCR assays. Cycle threshold (C ) values were descriptively compared.

Results: Based solely on mathematical modeling, the three PCR assays showed considerably different detection rates of T. whipplei DNA in stool samples (kappa 0.67 (95% confidence interval [0.60, 0.73])). Considering the calculated test characteristics, prevalence of 28.3% for group 1 and 5.0% for group 2 was estimated. Discordant test results were associated with later C values. The study did not validate the assays for the detection of T. whipplei in Whipple's disease and for diagnostic purposes since clinical specificity and sensitivity were not investigated.

Conclusions: In spite of the observed diagnostic uncertainty, PCR-based screening approaches can be used for epidemiological purposes and environmental samples to define the source and reservoir in resource-limited tropical settings if prevalence is calculated using diagnostic accuracy-adjusted methods.
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http://dx.doi.org/10.1111/tmi.13172DOI Listing
January 2019

Acrylic microparticles increase daptomycin intracellular and in vivo anti-biofilm activity against Staphylococcus aureus.

Int J Pharm 2018 Oct 25;550(1-2):372-379. Epub 2018 Aug 25.

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Electronic address:

Daptomycin (DAP) is a cyclic lipopeptide antibiotic with potential clinical application in orthopedic infections caused by staphylococci. However, it failed to eradicate Staphylococcus aureus in vitro, in intracellular infection studies, as well as in vivo in an experimental model of implant-associated biofilm infections. In this study, the antimicrobial effect of DAP encapsulated in poly(methyl methacrylate)-Eudragit (PMMA-EUD) microparticles (DAP-MPs) on intracellular S. aureus was evaluated in human osteoblast cells using fluorescence in situ hybridization (FISH) analysis. Encapsulated DAP was able to reduce the amount of intracellular S. aureus by 73% compared to blank microparticles (MPs). Then, the advantage of treating with DAP-MPs versus free DAP was evaluated in a murine model of implant-associated biofilm infection. Free DAP showed a >3 log decrease in planktonic and adherent bacteria but failed to eradicate adherent methicillin-resistant S. aureus (MRSA), whereas DAP-MPs showed a clearance of planktonic MRSA, significantly reduced adherent MRSA by more than 3 log and cured the infection in 60%. This was linked to the prolonged higher DAP concentration within the tissue cage fluid compared to free DAP. To our knowledge, this study provides the first evidence for the high intracellular and in vivo anti-biofilm efficacy of DAP-MPs to target staphylococcal infections.
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http://dx.doi.org/10.1016/j.ijpharm.2018.08.048DOI Listing
October 2018

Whipple's Disease: Diagnostic Value of rpoB Gene PCR from Peripheral Blood Mononuclear Cells.

Mol Diagn Ther 2018 08;22(4):459-469

Department for Gastroenterology, Infectious Diseases, and Rheumatology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203, Berlin, Germany.

Introduction: Chronic infection with Tropheryma whipplei, known as Whipple's disease (WD), classically affects the gastrointestinal tract, but any organ system may be affected, and isolated manifestations occur. Reliable diagnosis based on a combination of periodic acid-Schiff (PAS) staining, T. whipplei-specific immunohistochemistry (IHC), and polymerase chain reaction (PCR) from duodenal biopsies may be challenging in cases without classical gastrointestinal infection, so the need for additional diagnostic materials is urgent.

Objective: Our objective was to evaluate additional diagnostic possibilities for WD.

Methods: We analyzed samples from 20 patients with WD and 18 control subjects in a prospective observational pilot study. In addition to WD diagnosis by PAS staining, T. whipplei-specific IHC and PCR of duodenal or extra intestinal tissues, whole EDTA blood, peripheral blood mononuclear cells (PBMCs) and PBMC fractions enriched with or depleted of cluster of differentiation (CD)-14 cells were examined using T. whipplei rpoB gene PCR.

Results: Tropheryma whipplei DNA was detected in 35 of 60 (58.3%) preparations from 16 of 20 patients with WD, most of whom lacked gastrointestinal signs and characteristic PAS-positive duodenal macrophages.

Conclusion: This study provides evidence for the potential suitability of blood, particularly PBMCs, as material to assist in the diagnosis of WD via rpoB gene real-time PCR. Thus, PCR from blood preparations can be helpful for diagnostic decision making in atypical cases of WD.
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http://dx.doi.org/10.1007/s40291-018-0339-7DOI Listing
August 2018

Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus.

PLoS One 2018 13;13(4):e0195516. Epub 2018 Apr 13.

Department of Surgery, University Hospital of Bonn, Bonn, Germany.

Aim: To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus.

Methods: C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay.

Results: Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI.

Conclusion: Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195516PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5898729PMC
July 2018

Encapsulation in Polymeric Microparticles Improves Daptomycin Activity Against Mature Staphylococci Biofilms-a Thermal and Imaging Study.

AAPS PharmSciTech 2018 May 27;19(4):1625-1636. Epub 2018 Feb 27.

Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.

Eradication of Gram-positive biofilms is a critical aspect in implant-associated infection treatment. Although antibiotic-containing particulate carriers may be a promising strategy for overcoming biofilm tolerance, the assessment of their interaction with biofilms has not been fully explored. In the present work, the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (EUD) microparticles against methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive S. epidermidis biofilms was investigated using isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). The minimal biofilm inhibitory concentrations (MBIC) of MRSA biofilms, as determined by IMC, were 5 and 20 mg/mL for daptomycin- and vancomycin-loaded PMMA microparticles, respectively. S. epidermidis biofilms were less susceptible, with a MBIC of 20 mg/mL for daptomycin-loaded PMMA microparticles. Vancomycin-loaded microparticles were ineffective. Adding EUD to the formulation caused a 4- and 16-fold reduction of the MBIC values of daptomycin-loaded microparticles for S. aureus and S. epidermidis, respectively. FISH corroborated the IMC results and provided additional insights on the antibiofilm effect of these particles. According to microscopic analysis, only daptomycin-loaded PMMA-EUD microparticles were causing a pronounced reduction in biofilm mass for both strains. Taken together, although IMC indicated that a biofilm inhibition was achieved, microscopy showed that the biofilm was not eradicated and still contained FISH-positive, presumably viable bacteria, thus indicating that combining the two techniques is essential to fully assess the effect of microparticles on staphylococcal biofilms.
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http://dx.doi.org/10.1208/s12249-018-0974-7DOI Listing
May 2018

Detection of Coxiella burnetii in heart valve sections by fluorescence in situ hybridization.

J Med Microbiol 2018 Apr 20;67(4):537-542. Epub 2018 Feb 20.

Bundeswehr Institute of Microbiology, Munich, Germany.

Purpose: Infective endocarditis is a severe and potentially fatal disease. Nearly a third of all cases remain culture-negative, making a targeted and effective antibiotic therapy of patients challenging. In the past years, fluorescence in situ hybridization (FISH) has proven its value for the diagnosis of infective endocarditis, particularly when it is caused by fastidious bacteria. To increase the number of infective endocarditis causing agents, which can be identified by FISH, we designed and optimized a FISH-probe for the specific detection of Coxiella burnetii in heart valve tissue.

Methodology: Even with specific probes the detection and identification of bacteria can be complicated by the high autofluorescence due to calcification of the analysed tissue. To overcome this problem, we developed a protocol to detect C. burnetii by hybridizing, stripping and reprobing the identical section with different species-specific probes repeatedly.Results/Key findings. The newly designed specific FISH probe and the developed protocol exemplarily allowed us to unequivocally identify C. burnetii in tissue sections of a patient with infective endocarditis.

Conclusion: This method provides an add-on to existing protocols for the unambiguous diagnosis of bacteria directly within tissues or other difficult tissue samples in cases with small sample size and limited sections.
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http://dx.doi.org/10.1099/jmm.0.000704DOI Listing
April 2018

Life on the driveline: Molecular detection and fluorescence in situ hybridization-based visualization of microbial species in patients with left ventricular assist devices.

J Heart Lung Transplant 2018 01 30;37(1):163-166. Epub 2017 Sep 30.

DZHK (German Centre for Cardiovascular Research), partner site Berlin, Germany; Biofilm Center, German Heart Center Berlin, Berlin, Germany.

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http://dx.doi.org/10.1016/j.healun.2017.09.023DOI Listing
January 2018

serotype 9 endocarditis and subsequent severe meningitis in a growing pig despite specific bactericidal humoral immunity.

JMM Case Rep 2017 May 3;4(5):e005093. Epub 2017 May 3.

Institute for Bacteriology and Mycology, Centre for Infectious Diseases, Faculty of Veterinary Medicine, University Leipzig, Germany.

Introduction: . Meningitis and endocarditis are common pathologies of infections in pigs and humans. serotype 9 strains contribute substantially to health problems in European pig production, and immune prophylaxis against this serotype is very difficult.

Case Presentation: . We report the clinical course and histopathological picture of a 10-week-old growing pig following experimental intravenous infection with serotype 9. The piglet showed rapid onset of severe clinical signs of meningitis 11 days post-intravenous challenge following prime-booster vaccination. Histopathological findings revealed a diffuse fibrinosuppurative leptomeningitis. Additionally, a polyphasic with numerous bacterial colonies was diagnosed. Bacteriological culture of the brain and the mitral valve confirmed association with the challenge strain. However, virulent serotype 2 and 9 strains were killed in the blood of this piglet prior experimental infection.

Conclusion: . Endocarditis induced by infection might develop and persist despite the presence of high specific bactericidal activity in the blood. Severe leptomeningitis is a putative sequela of such an endocarditis.
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http://dx.doi.org/10.1099/jmmcr.0.005093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630962PMC
May 2017

Reaction-diffusion theory explains hypoxia and heterogeneous growth within microbial biofilms associated with chronic infections.

NPJ Biofilms Microbiomes 2016 22;2:16012. Epub 2016 Jun 22.

Biofilmcenter, German Heart Institute Berlin, Berlin, Germany.

Reaction-diffusion models were applied to gain insight into the aspects of biofilm infection and persistence by comparing mathematical simulations with the experimental data from varied bacterial biofilms. These comparisons, including three systems and two clinical investigations of specimens examined , underscored the central importance of concentration gradients of metabolic substrates and the resulting physiological heterogeneity of the microorganisms. Relatively simple one-dimensional and two-dimensional (2D) models captured the: (1) experimentally determined distribution of specific growth rates measured in cells within sputum from cystic fibrosis patients; (2) pattern of relative growth rate within aggregates of streptococcal biofilm harboured in an endocarditis vegetation; (3) incomplete penetration of oxygen into a biofilm under conditions of exposure to ambient air and also pure oxygen; (4) localisation of anabolic activity around the periphery of cell clusters formed in a flow cell and attribution of this pattern to iron limitation; (5) very low specific growth rates, as small as 0.025 h, in the interior of cell clusters within a biofilm in a complex 2D domain of variable cell density.
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http://dx.doi.org/10.1038/npjbiofilms.2016.12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515263PMC
June 2016

Effects of Diode Laser, Gaseous Ozone, and Medical Dressings on Biofilms in the Root Canal Ex Vivo.

Biomed Res Int 2017 10;2017:6321850. Epub 2017 Apr 10.

Department of Operative Dentistry and Preventive Dentistry, CharitéCentrum 3, Charité-Universitätsmedizin Berlin, Aßmannshauser Str. 4-6, 14197 Berlin, Germany.

The objective was to compare the antibacterial effects of adjunctive disinfection using diode laser and gaseous ozone compared to the medical dressings calcium hydroxide (Ca(OH)) and chlorhexidine gel (CHX-Gel) on biofilms in human root canals ex vivo. Root canals of 180 human extracted teeth were infected by and divided into 3 main groups (G): G1, control; G2, instrumentation and irrigation using 0.9% NaCl; G3, instrumentation and irrigation using 1% NaOCl. In each main group, the following treatments were applied: gaseous ozone, diode laser, and medical dressings of Ca(OH) or CHX-Gel for 7 days ( = 15). Reduction of colony forming units (CFUs) inside the root canal of planktons and frequencies of adherent bacteria after treatment were calculated. Bacterial reduction was significantly affected by the irrigation protocol ( < 0.0005) and the disinfection method ( < 0.0005), and a significant interaction between both factors could be observed ( < 0.0005; ANOVA). In G3 (instrumentation using 1% NaOCl), no significant effect of disinfection methods could be demonstrated on planktonic bacteria ( = 0.062; ANOVA) and frequencies of adherent bacteria ( > 0.05; chi-square test). Instrumentation and irrigation using NaOCl combined with ozone or laser application resulted in comparable bacterial reduction on to the application of medical dressings.
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http://dx.doi.org/10.1155/2017/6321850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439256PMC
March 2018

Identification of Tp0751 (Pallilysin) as a Treponema pallidum Vascular Adhesin by Heterologous Expression in the Lyme disease Spirochete.

Sci Rep 2017 05 8;7(1):1538. Epub 2017 May 8.

Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.

Treponema pallidum subsp. pallidum, the causative agent of syphilis, is a highly invasive spirochete pathogen that uses the vasculature to disseminate throughout the body. Identification of bacterial factors promoting dissemination is crucial for syphilis vaccine development. An important step in dissemination is bacterial adhesion to blood vessel surfaces, a process mediated by bacterial proteins that can withstand forces imposed on adhesive bonds by blood flow (vascular adhesins). The study of T. pallidum vascular adhesins is hindered by the uncultivable nature of this pathogen. We overcame these limitations by expressing T. pallidum adhesin Tp0751 (pallilysin) in an adhesion-attenuated strain of the cultivable spirochete Borrelia burgdorferi. Under fluid shear stress representative of conditions in postcapillary venules, Tp0751 restored bacterial-vascular interactions to levels similar to those observed for infectious B. burgdorferi and a gain-of-function strain expressing B. burgdorferi vascular adhesin BBK32. The strength and stability of Tp0751- and BBK32-dependent endothelial interactions under physiological shear stress were similar, although the mechanisms stabilizing these interactions were distinct. Tp0751 expression also permitted bacteria to interact with postcapillary venules in live mice as effectively as BBK32-expressing strains. These results demonstrate that Tp0751 can function as a vascular adhesin.
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http://dx.doi.org/10.1038/s41598-017-01589-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431505PMC
May 2017

Microbiological diagnosis of device-related biofilm infections.

APMIS 2017 Apr;125(4):289-303

Medical Biotechnology, Danish Technological Institute, Aarhus, Denmark.

Medical device-related infections cause undue patient distress, increased morbidity and mortality and pose a huge financial burden on healthcare services. The pathogens are frequently distributed heterogeneously in biofilms, which can persist without being effectively cleared by host immune defenses and antibiotic therapy. At present, there is no 'gold standard' available to reveal the presence of device-related biofilm infections. However, adequate sample collection and logistics, standardised diagnostic methods, and interpretation of results by experienced personnel are important steps in efficient diagnosis and treatment of these infections. The focus of this mini review is on prosthethic joint and cardiovascular implantable device infections, which exemplify permanent devices that are placed in a sterile body site. These device-related infections represent some of the most challenging in terms of both diagnosis and treatment.
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http://dx.doi.org/10.1111/apm.12676DOI Listing
April 2017

Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis.

Anaerobe 2017 Oct 12;47:25-32. Epub 2017 Mar 12.

Biofilmcenter, Deutsches Herzzentrum Berlin, Berlin, Germany; Former German Consultant Laboratory for Treponema Identification, Charité - Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men).
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http://dx.doi.org/10.1016/j.anaerobe.2017.03.012DOI Listing
October 2017

Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review.

Crit Rev Microbiol 2017 May 27;43(3):263-293. Epub 2017 Jan 27.

e Institute for Medical Microbiology, Justus-Liebig-University Giessen , Giessen , Germany.

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.
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http://dx.doi.org/10.3109/1040841X.2016.1169990DOI Listing
May 2017

A Novel Mouse Model of Staphylococcus aureus Vascular Graft Infection: Noninvasive Imaging of Biofilm Development in Vivo.

Am J Pathol 2017 Feb 11;187(2):268-279. Epub 2017 Jan 11.

Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.

Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development.
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http://dx.doi.org/10.1016/j.ajpath.2016.10.005DOI Listing
February 2017

Rapid molecular diagnosis of infective aortic valve endocarditis caused by Coxiella burnetii.

Infection 2016 Dec 23;44(6):813-817. Epub 2016 Jun 23.

Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, Charité-University Medicine Berlin, Charitéplatz 1, 10117, Berlin, Germany.

We describe a case of Q-fever endocarditis with severe destruction of the aortic valve with perivalvular abscess formation and cardiac failure. The patient needed urgent operative treatment and postoperative critical care. All specimens sent for microbiological examination were negative. Molecular analysis, including fluorescence in situ hybridization of aortic valve tissue combined with PCR and sequencing, led to the correct diagnosis and to appropriate anti-infective treatment. The patient subsequently recovered from complex cardiovascular surgery. This is the first report on Q-fever endocarditis that was rapidly diagnosed using these methods.
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http://dx.doi.org/10.1007/s15010-016-0916-9DOI Listing
December 2016