Pathogen spectrum of community acquired pneumonia in people living with HIV (PLWH) in the German CAPNETZ-Cohort.

OBJECTIVES: The objective of this study was to identify the pathogen spectrum of community acquired pneumonia in people living with HIV (PLWH), and to compare it with a matched HIV negative group in order to reassess therapeutic strategies for PLWH. METHODS: Seventy-three (n = 73) PLWH (median CD4 3-6 months before CAP: 515/µl; SD 309) with community acquired pneumonia (CAP) were matched with 218 HIV-negative CAP controls in a prospective study design. Pathogen identifications used blood culture, samples from the upper and lower respiratory tract (culture and multiplex PCR) and urinary pneumococcal and legionella antigen test. RESULTS: Although the vaccination rate among PLWH with CAP was significantly higher (pneumococcal vaccination: 27.4 vs. 8.3%, p < 0.001; influenza vaccination: 34.2 vs. 17.4%, p = 0.009), pneumococci were found most frequently as pathogen among both PLWH (n = 19/21.3%) and controls (n = 34/17.2%; p = 0.410), followed by Haemophilus influenzae (PLWH, n = 12/13.5%, vs. controls, n = 25 / 12.6%; p = 0.850). Staphylococcus aureus was found equally in 20.2 and 19.2% in PLWH and controls, but infection or colonization could not be distinguished. Mortality during 6-month follow-up was significantly higher for PLWH (5/73, or 6.8%) versus controls (3/218, or 1.4%), however with lower case numbers than previously reported. Typical HIV-associated pathogens such as Pneumocystis jirovecii were found only exceptionally. CONCLUSIONS: Our study underscores the persistent clinical burden of CAP for PLWH. From pathogen perspective, empirical antibiotic treatment for CAP in PLWH on antiretroviral therapy should cover pneumococci and Haemophilus influenzae and may be adopted from valid common recommendations.

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.

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.

Evaluation of a multiplex PCR screening approach to identify community-acquired bacterial co-infections in COVID-19: a multicenter prospective cohort study of the German competence network of community-acquired pneumonia (CAPNETZ).

PURPOSE: Thorough knowledge of the nature and frequency of co-infections is essential to optimize treatment strategies and risk assessment in cases of coronavirus disease 2019 (COVID-19). This study aimed to evaluate the multiplex polymerase chain reaction (PCR) screening approach for community-acquired bacterial pathogens (CABPs) at hospital admission, which could facilitate identification of bacterial co-infections in hospitalized COVID-19 patients. METHODS: Clinical data and biomaterials from 200 hospitalized COVID-19 patients from the observational cohort of the Competence Network for community-acquired pneumonia (CAPNETZ) prospectively recruited between March 17, 2020, and March 12, 2021 in 12 centers in Germany and Switzerland, were included in this study. Nasopharyngeal swab samples were analyzed on hospital admission using multiplex real-time reverse transcription (RT)-PCR for a broad range of CABPs. RESULTS: In total of 200 patients Staphylococcus aureus (27.0%), Haemophilus influenzae (13.5%), Streptococcus pneumoniae (5.5%), Moraxella catarrhalis (2.5%), and Legionella pneumophila (1.5%) were the most frequently detected bacterial pathogens. PCR detection of bacterial pathogens correlated with purulent sputum, and showed no correlation with ICU admission, mortality, and inflammation markers. Although patients who received antimicrobial treatment were more often admitted to the ICU and had a higher mortality rate, PCR pathogen detection was not significantly related to antimicrobial treatment. CONCLUSION: General CABP screening using multiplex PCR with nasopharyngeal swabs may not facilitate prediction or identification of bacterial co-infections in the early phase of COVID-19-related hospitalization. Most patients with positive PCR results appear to be colonized rather than infected at that time, questioning the value of routine antibiotic treatment on admission in COVID-19 patients.

Caesarean Section. Guideline of the DGGG, OEGGG and SGGG (S3-Level, AWMF Registry No. 015/084, June 2020).

Purpose This is an official S3-guideline of the German Society of Gynaecology and Obstetrics (DGGG), the Austrian Society of Gynaecology and Obstetrics (ÖGGG) and the Swiss Society of Gynaecology and Obstetrics (SGGG). The guideline contains evidence-based information and recommendations on indications, complications, methods and care associated with delivery by caesarean section for all medical specialties involved as well as for pregnant women. Methods This guideline has adapted information and recommendations issued in the NICE Caesarean Birth guideline. This guideline also considers additional issues prioritised by the Cochrane Institute and the Institute for Research in Operative Medicine (IFOM). The evaluation of evidence was based on the system developed by the Scottish Intercollegiate Guidelines Network (SIGN). A multi-part nominal group process moderated by the AWMF was used to compile this S3-level guideline. Recommendations Recommendations on consultations, indications and the process of performing a caesarean section as well as the care provided to the mother and neonate were drawn up.

The impact of the SARS-CoV-2 pandemic on the prevalence of respiratory tract pathogens in patients with community-acquired pneumonia in Germany.

We show a shift in the prevalence of respiratory viral pathogens in community-acquired pneumonia patients during the COVID-19 pandemic. Our data support the efficiency of non-pharmaceutical interventions on virus circulation except for rhinoviruses. The consequences of an altered circulation on subsequent winter seasons remain unclear and support the importance of systematic virological surveillance.

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.

Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αß-defensins (Csαß-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

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.

Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.

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.

Microbiological diagnosis and molecular typing of Legionella strains during an outbreak of legionellosis in Southern Germany.

An explosive outbreak of Legionnaires' disease with 64 reported cases occurred in Ulm/Neu-Ulm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 min reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection.

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae.

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammalsO-mannosylation is the only type ofO-glycosylation. In an essential step toward the full understanding of proteinO-mannosylation we mapped theO-mannose glycoproteome in baker's yeast. Taking advantage of anO-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500O-glycoproteins from all subcellular compartments for which over 2300O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized proteinO-mannosyltransferases. We find thatO-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed thatO-mannosylation is favored in unstructured regions and ß-strands. Furthermore,O-mannosylation is impeded in the proximity ofN-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and theirO-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types ofO-glycosylation from yeast to mammals.

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.

Subtyping of the Legionella pneumophila "Ulm" outbreak strain using the CRISPR-Cas system.

In 2009/2010 an outbreak of Legionnaires' disease with 64 cases including four fatalities took place in the city of Ulm/Neu-Ulm in Germany. L. pneumophila serogroup 1, mAb type Knoxville, sequence type (ST) 62 was identified as the epidemic strain. This strain was isolated from eight patients and from a cooling tower in the city of Ulm. Based on whole genome sequencing data from one patient strain, we identified an Lvh type IV secretion system containing a CRISPR-Cas system. The CRISPR sequence contains 38 spacer DNA sequences. We used these variable DNA spacers to further subtype the outbreak strain as well as six epidemiologically unrelated strains of CRISPR-Cas positive ST62 strains isolated at various regions in Germany. The first 12 spacer DNAs of eight patient isolates and three environmental isolates from the suspected source of infection were analyzed and found to be identical. Spacer DNAs were identified in further six epidemiologically unrelated patient isolates of L. pneumophila of ST62 in addition to the 12 "core" spacers. The presence of new spacer DNAs at the 5' site downstream of the first repeat indicates that these CRISPR-Cas systems seem to be functional. PCR analysis revealed that not all L. pneumophila sg1 ST62 strains investigated exhibited a CRISPR-Cas system. In addition, we could demonstrate that the CRISPR-Cas system is localized on a genomic island (LpuGI-Lvh) which can be excised from the chromosome and therefore may be transferable horizontally to other L. pneumophila strains.

Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/ß-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.