Differences in oxazolidinone resistance mechanisms and small colony variants emergence of Staphylococcus aureus induced in an in vitro resistance development model.

Invasive Staphylococcus aureus infections are associated with a high burden of disease, case fatality rate and healthcare costs. Oxazolidinones such as linezolid and tedizolid are considered potential treatment choices for conditions involving methicillin resistance or penicillin allergies. Additionally, they are being investigated as potential inhibitors of toxins in toxin-mediated diseases. In this study, linezolid and tedizolid were evaluated in an in vitro resistance development model for induction of resistance in S. aureus. Whole genome sequencing was conducted to elucidate resistance mechanisms through the identification of causal mutations. After inducing resistance to both linezolid and tedizolid, several partially novel single nucleotide variants (SNVs) were detected in the rplC gene, which encodes the 50S ribosome protein L3 in S. aureus. These SNVs were found to decrease the binding affinity, potentially serving as the underlying cause for oxazolidinone resistance. Furthermore, in opposite to linezolid we were able to induce phenotypically small colony variants of S. aureus after induction of resistance with tedizolid for the first time in literature. In summary, even if different antibiotic concentrations were required and SNVs were detected, the principal capacity of S. aureus to develop resistance to oxazolidinones seems to differ between linezolid and tedizolid in-vivo but not in vitro. Stepwise induction of resistance seems to be a time and cost-effective tool for assessing resistance evolution. Inducted-resistant strains should be examined and documented for epidemiological reasons, if MICs start to rise or oxazolidinone-resistant S. aureus outbreaks become more frequent.

18FDG-PET/CT-Scans and Biomarker Levels Predicting Clinical Outcome in Patients with Alveolar Echinococcosis-A Single-Center Cohort Study with 179 Patients.

Background: Alveolar echinococcosis (AE) is a severe larval tapeworm infection with a variable clinical course of the disease. Reliable imaging techniques and biomarkers are needed to predict the course of the disease. Methods: 179 AE patients that received PET/CT scans between 2008 and 2012 were retrospectively included. From stored blood samples taken on the day of the scan, levels of IgE, parasite-specific serology, amyloid A, C-reactive protein, soluble interleukin 2 receptor, cytokeratin fragments, eosinophilic cell count, and eosinophil cationic protein were measured. Additionally, the current clinical outcome (cured, stable, or progressive disease) after a median duration of 8 years after baseline examination was assessed. Ultimately, an ordinal logistic regression was conducted to evaluate which imaging parameters and biomarkers independently influence the clinical outcome. Results: In general, patients in need of medical treatment or with progressive disease, advanced PNM stages, and positive PET/CT scans exhibited higher levels of the respective biomarkers. However, only the parasite-specific serological markers and total IgE levels differed significantly between clinical groups, WHO PNM stages, and the results of the PET/CT scan. In the multivariate analysis, PET/CT results were a strong predictor of the clinical outcome (OR 8.908, 95%CI 3.019-26.285; p < 0.001), and age at baseline was a moderate predictor (OR 1.031, 95%CI 1.003-1.060; p = 0.029). Conclusions: The PET/CT scan is, preferably in combination with parasite-specific serology and IgE levels, a valuable tool in the clinical management of AE and is able to predict the course of the disease.

Clearing Chlamydia abortus infection in epithelial cells and primary human macrophages by use of antibiotics and the MDM2-p53-inhibitor nutlin-3.

Chlamydia (C.) abortus is an emerging zoonotic pathogen. Since data on its antimicrobial susceptibility are lacking, we aimed to determine minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) for azithromycin and doxycycline in HeLa-cells and primary human macrophages (M1). We also examined the MDM2-p53-inhibitor nutlin-3, an anti-infective imidazoline analog. Azithromycin and doxycycline demonstrated MICs and MBCs equal or below their peak serum concentrations (PSC) after standard dosing in both cell types. While doxycycline exhibited an MIC 64-fold and an MBC 4-fold below its PSC in HeLa-cells, the MIC of azithromycin was 4-fold below, the MBC equal to the PSC. However, azithromycin revealed lower MBCs in M1. The pharmacological advantage of azithromycin accumulation in phagocytes and their role as chlamydial reservoirs remain uncertain. However, our data suggest possible therapeutic advantages of doxycycline in epithelial cells and we provide first evidence for an anti-C. abortus effect of nutlin-3 in M1.

Initial Risk Assessment in Patients with Alveolar Echinococcosis-Results from a Retrospective Cohort Study.

Background: Alveolar echinococcosis (AE) is a potentially lethal parasitosis with a broad spectrum of disease dynamics in affected patients. To guide clinical management, we assessed initial prognostic factors for both progressive and controlled AE based on initial staging. Methods: A retrospective cohort study was conducted, examining 279 patients assigned to different clinical groups: cured, stable with and without the need for benzimidazole treatment, and progressive disease. Univariate analysis compared demographic and clinical variables. Significant variables were subsequently entered into two separate logistic regression models for progressive and controlled disease. Results: Based on the multivariate analysis, a large AE lesion (OR = 1.02 per millimetre in size; 95%CI 1.004-1.029), PNM staging (OR = 2.86; 95%CI 1.384-5.911) and especially the involvement of neighbouring organs (OR = 3.70; 95%CI 1.173-11.653) remained significant risk factors for progressive disease. A negative Em2+ IgG (OR = 0.25; 95%CI 0.072-0.835) and a small AE lesion (OR = 0.97; 95%CI 0.949-0.996) were significant protective factors. Conclusions: Patients with large lesions and advanced stages should be monitored closely and most likely require long-term treatment with benzimidazoles if curative resection is not feasible. Patients with small lesions and negative Em2+ IgG seem able to control the disease to a certain extent and a less strict treatment regimen might suffice.

Evaluation of Serological Markers in Alveolar Echinococcosis Emphasizing the Correlation of PET-CTI Tracer Uptake with RecEm18 and Echinococcus-Specific IgG.

Human alveolar echinococcosis (AE), which is caused by the cestode Echinococcus (E.) multilocularis, is an epidemiologically relevant issue in modern medicine and still poses a diagnostic and therapeutic challenge. Since diagnosis mainly relies on imaging procedures and serological testing, we retrospectively and comparatively analyzed the performance of an Echinococcus IgG screening ELISA, whole serum IgE, and two specific confirmatory ELISA platforms using the defined E. multilocularis antigens Em2-Em18 (Em2+) and recombinant Em18 (recEm18). With special emphasis on the clinical usefulness of recEm18, we correlated the laboratory results with clinical characteristics and imaging findings in a large and well-characterized cohort of N = 124 AE patients, who were followed over several years after either surgical plus subsequent pharmacological treatment or pharmacotherapy alone. All patients had routinely received PET-CTI every two years. Our data reveal strong correlations for both Echinococcus IgG and recEm18 with tracer uptake in PET-CTI and parasitic lesion size and number, suggesting additional clinical usefulness of recEm18 for certain constellations only, while IgG and Em2+ still appear reasonable and sensitive screening methods for initial diagnosis of AE. With this study, we aim to contribute to further optimizing medical care of AE patients. For instance, it might be reasonable to consider the replacement of some PET-CTI follow-ups by imaging procedures with less radiation exposure or serological means alone. Further studies that clarify the correlation of serological markers with ultrasound criteria might be particularly useful, and further retrospective as well as prospective investigations are justified in this context.

First case of fatal Rhizomucor miehei endocarditis in an immunocompromised patient.

Rhizomucor miehei is a cause of bovine mycotic abortion and mastitis and has rarely been described in human disease. Here, we report the first isolation of R. miehei from native mitral valve tissue in a fatal case of endocarditis that substantiates its pathogenic potential. Apart from morphological criteria, molecular methods were a cornerstone for definite diagnosis.

The Putative Type III Secreted Chlamydia abortus Virulence-Associated Protein CAB063 Targets Lamin and Induces Apoptosis.

Since intracellular survival of all chlamydiae depends on the manipulation of the host cell through type III secreted effector proteins, their characterization is crucial for the understanding of chlamydial pathogenesis. We functionally characterized the putative type III secreted Chlamydia abortus protein CAB063, describe its intracellular localization and identified pro- and eukaryotic binding partners. Based on an experimental infection model and plasmid transfections, we investigated the subcellular localization of CAB063 by immunofluorescence microscopy, immunoelectron microscopy, and Western blot analysis. Pro- and eukaryotic targets were identified by co-immunofluorescence, co-immunoprecipitation, and mass spectrometry. Transmission electron microscopy and flow cytometry were used for morphological and functional investigations on host cell apoptosis. CAB063 localized in the nuclear membrane of the host cell nucleus and we identified the chaperone HSP70 and lamin A/C as pro- and eukaryotic targets, respectively. CAB063-dependent morphological alterations of the host cell nucleus correlated with increased apoptosis rates of infected and CAB063-transfected cells. We provide evidence that CAB063 is a chaperone-folded type III secreted C. abortus virulence factor that targets lamin thereby altering the host cell nuclear membrane structure. This process may be responsible for an increased apoptosis rate at the end of the chlamydial developmental cycle, at which CAB063 is physiologically expressed.

Analysis of humoral immune responses to recombinant Chlamydia pneumoniae antigens.

OBJECTIVES: Chlamydia pneumoniae is a difficult to diagnose respiratory pathogen. This study was performed to systematically characterize humoral immune responses to selected C. pneumoniae antigens in order to provide novel serodiagnostic perspectives for clinical and epidemiological issues. METHODS: Based on a literature search, gene library screening, and serological proteome analysis, 15 immunogenic surface-associated, virulence-associated, and hypothetical C. pneumoniae antigens were selected, recombinantly expressed, and lined on a nitrocellulose strip. Specific IgM and IgG reactivity was measured in a total of 172 PCR- and micro-immunofluorescence testing (MIF)-characterized serum samples from patients with respiratory infections. A theoretical model was conceived to approximate a putative course of C. pneumoniae antigen expression and assess the potential of early and late antigens. RESULTS: While surface antigens performed poorly, the virulence-associated TARP was a reliable antigen for IgM detection, with a sensitivity of 80.0% and a diagnostic specificity of 90.2%. The hypothetical protein YwbM proved powerful for IgG detection with MIF-correlative sensitivities of up to 94.4% and a diagnostic specificity of 95.1%. CONCLUSIONS: This study provides new insights into antibody profiles to immunogenic proteins in C. pneumoniae infection. The study findings offer antigen candidates for more reliable and standardized serological investigations of C. pneumoniae infections, including studies on seroprevalence and epidemiology.

Pulmonary Campylobacter concisus infection in an immunocompromised patient with underlying mucormycosis.

Campylobacter concisus is a rarely encountered agent of human infection. The first isolation of C. concisus from a pulmonary abscess in an immunocompromised patient who underwent allogeneic stem cell transplantation is reported here. This unusual case demonstrates the pathogenic potential of this bacterium and outlines species-immanent difficulties in gaining a reliable diagnosis. Molecular methods were a cornerstone for definite identification of the organism grown on anaerobic culture from surgically excised tissue. Antimicrobial susceptibility testing revealed unusual quinolone and macrolide resistance, and therefore antimicrobial therapy was based on ß-lactam antibiotics.

KPC-2 carbapenemase-producing Pseudomonas aeruginosa reaching Germany.

Background: Antimicrobial resistance due to carbapenemase expression poses a worldwide threat in healthcare. Inter-genus exchange of genetic information is of utmost importance in this context. Objectives: Here, to the best of our knowledge, we describe the first detection and characterization of a KPC-2-producing Pseudomonas aeruginosa in Germany. Methods: Characterization of the isolate was performed using MALDI-TOF MS, automated microdilution and MLST. Carbapenemase detection was performed using phenotypic and genotypic assays. The blaKPC-2-carrying plasmid was transformed into Escherichia coli NEB® 10-beta. The purified plasmid DNA was sequenced using the Illumina technique. Results: The isolate expressed ST235 and was resistant to carbapenems. Antimicrobial susceptibility testing revealed colistin to be the only antimicrobial agent active in vitro. The blaKPC-2 gene was located on a replicon type lncHI1 plasmid as part of Tn4401. Conclusions: The first detection (to the best of our knowledge) of plasmid-encoded KPC-2 in P. aeruginosa in Germany may point to a currently underestimated spread of carbapenemases among clinically relevant Gram-negative bacteria. Here, to the best of our knowledge, we also provide the first report of blaKPC-2 associated with the IncHI1 plasmid.

Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

Early prosthetic valve endocarditis caused by Corynebacterium kroppenstedtii.

Corynebacterium (C.) kroppenstedtii is a rarely detected agent of bacterial infections in humans. Here, we describe the first case of prosthetic valve endocarditis caused by C. kroppenstedtii. Application of molecular methods using surgically excised valve tissue was a cornerstone for the establishment of the microbiological diagnosis, which is crucial for targeted antimicrobial treatment.