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.

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.

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.

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.

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.

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.

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.

Identification of lymphogranuloma venereum-associated Chlamydia trachomatis serovars by fluorescence in situ hybridisation--a proof-of-principle analysis.

We describe a proof-of-principle evaluation of a fluorescence in situ hybridisation (FISH) procedure to identify Chlamydia trachomatis serovars L1-L3, the causative agents of lymphogranuloma venereum, in cell cultures based on newly designed DNA probes. Rapid and easy-to-perform FISH could facilitate the diagnosis of lymphogranuloma venereum without nucleic acid amplification or serotyping, but requires broader evaluation studies, for example, in tropical high-endemicity regions.

Resurgence of common respiratory viruses in patients with community-acquired pneumonia (CAP)-A prospective multicenter study.

BACKGROUND: Community-acquired pneumonia (CAP) is a major global cause of death and hospitalization. Bacteria or community-acquired viruses (CARVs) cause CAP. COVID-19 associated restrictions effectively reduced the circulation of CARVs. OBJECTIVES: The aim of this study was to analyze the proportion of CARVs in adult patients with CAP from mid-2020 to mid-2023. Specifically, we aimed to compare the rate of influenza virus, SARS-CoV-2, and RSV detections in patients aged 18-59 years and ≥60 years. STUDY DESIGN: We analyze the proportion of 21 community-acquired respiratory viruses (CARVs) and three atypical bacteria (Bordetella pertussis, Legionella pneumophila, and Mycoplasma pneumoniae) in nasopharyngeal swab samples using molecular multiplex methods within the prospective, multicentre, multinational study of the German study Group CAPNETZ. We used stringent inclusion criteria throughout the study. RESULTS: We identified CARVs in 364/1,388 (26.2 %) patients. In detail, we detected SARS-CoV-2 in 210/1,388 (15.1 %), rhino-/enterovirus in 64/1,388 (4.6 %), influenza virus in 23/1,388 (1.6 %) and RSV in 17/1,388 (1.2 %) of all patients. We detected RSV and influenza more frequently in patients ≥60 years, especially in 22/23 compared to the previous season. None of the atypical bacteria were detected. CONCLUSIONS: Beginning in 2023, we demonstrate a re-emergence of CARVs in CAP patients. Effective vaccines or specific antiviral therapies for more than two thirds of the detected viral infections are currently available. High detection rates of vaccine-preventable viruses in older age groups support targeted vaccination campaigns.

Fluorescence in situ hybridization (FISH) for rapid identification of Salmonella spp. from agar and blood culture broth--an option for the tropics?

BACKGROUND: Salmonella enterica is an important cause of diarrhea with the potential to cause systemic infection including sepsis, particularly in the tropics. Sepsis in particular requires quick and reliable identification to allow a rapid optimization of antibiotic therapy. We describe the establishment and evaluation of fluorescence in situ hybridization (FISH) as a rapid and easy-to-perform molecular identification procedure from agar and blood culture broths. METHODS: Two newly developed FISH probes with specificity for Salmonella spp. were evaluated with 10 reference strains, 448 clinical isolates of Gram-negative bacteria from Germany and Ghana including 316 Salmonella spp. strains, and 39 environmental Salmonella spp. isolates from rivers and streams in Ghana. One FISH probe was further tested with 207 pre-incubated blood culture broths from Germany with Gram-negative rod-shaped bacteria in Gram stain. RESULTS: Evaluation of the newly designed FISH probes demonstrated sensitivity of 99.2% and specificity of 98.4% for clinical isolates, sensitivity of 97.4% for environmental Salmonella spp. isolates, and sensitivity of 100% and specificity of 99.5% for blood culture materials. CONCLUSIONS: FISH proved to be highly reliable for a rapid identification of Salmonella spp. directly from pre-incubated blood culture broths as well as after growth on agar. The inexpensive and easy-to-perform procedure is particularly suitable for resource-limited areas where more sophisticated procedures are not available.

Rapid identification of Leishmania spp. in formalin-fixed, paraffin-embedded tissue samples by fluorescence in situ hybridization.

OBJECTIVE: To describe and validate fluorescence in situ hybridization (FISH), a new method of Leishmania spp. identification. FISH allows for a rapid detection of target organisms by specific binding of fluorescently labelled oligonucleotide probes to ribosomal RNA. METHODS: Two genus-specific, fluorescently labelled Leishmania spp. FISH probes were designed and evaluated with a panel of 18 Leishmania spp. and six Trypanosoma spp. including well-defined strains and clinical isolates. In addition, the FISH probes were tested in comparison with Giemsa staining in formalin-fixed, paraffin-embedded tissues of five mice that had been artificially infected with Leishmania major strains, leading to concordant results. Finally, 11 tissue samples of patients with cutaneous leishmaniasis, four tissue samples of patients with visceral leishmaniasis, and one native bone marrow sample of a patient with visceral leishmaniasis were analysed with FISH and Giemsa staining. RESULTS: Concordant results were achieved by FISH and Giemsa staining in 15/16 specimens. CONCLUSION: This analysis provides proof of principle that FISH is a suitable method for the rapid and easy detection of Leishmania spp. in formalin-fixed, paraffin-embedded tissue samples. Because of the good contrast of Leishmania spp. in tissue, FISH facilitates the identification of these organisms in tissue samples even by less experienced investigators.

Evaluation of fluorescence in situ hybridisation (FISH) for the identification of Candida albicans in comparison with three phenotypic methods.

Severe Candida infections are increasing and are associated with considerable morbidity and mortality. Rapid and accurate differentiation of Candida albicans from non-C. albicans species is essential for therapeutic decisions. We therefore developed a fluorescence in situ hybridisation (FISH) assay comprising previously described probes and a newly designed specific C. albicans probe/competitor probe combination. The FISH probes were first evaluated using 99 selected fungal strains covering 31 species, and a specificity between 96% and 100% and a sensitivity of 100%. The FISH assay was then applied to 110 clinical isolates in parallel with API32C, the chromogenic Candida ID agar, and determination of filamentous colony morphology. All tests produced highly reliable results. However, the Candida ID agar misidentified Candida dubliniensis as C. albicans. Determination of filamentous colony morphology allowed 100% reliable identification of C. albicans, but took 48 h. FISH allowed identification of clinical C. albicans isolates within 3 h with a sensitivity and specificity of 100%. FISH was additionally applied to 48 blood cultures showing yeasts in the Gram stain and correctly identified all 33 cases of C. albicans.

Rapid identification of yeast by fluorescence in situ hybridisation from broth and blood cultures.

Candida species including species other than Candida albicans are of importance as causative agents of sepsis in intensive-care units, requiring prompt initiation of targeted therapy. While fluconazole is usually active against Candida albicans, non-Candida albicans species often require more sophisticated approaches. A rapid species diagnosis is therefore desirable and can be provided by fluorescence in situ hybridisation (FISH). However, broad evaluation studies of described probes are largely lacking and the probe panel that has been described is incomplete. As an addition to previously described C. albicans FISH probes, we evaluated published DNA probes for C. glabrata and C. krusei, as well as newly designed DNA probes for C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Crypotococcus neoformans and a group of intrinsically fluconazole-resistant Candida species for FISH with 22 reference strains, 23 well-characterised laboratory control strains, 169 isolates from clinical samples and 48 blood cultures. Sensitivity and specificity of >99% were demonstrated for all evaluated species-specific probes, whereas the probe that binds to a heterogeneous group of intrinsically fluconazole-resistant Candida species correctly identified eight of nine fluconazole-resistant clinical isolates. FISH yielded reliable results using the classical FISH procedure as well as a recently described slide chamber-based method. Given this good sensitivity and specificity, FISH may be applied for rapid identification of yeast in screening analyses, thus giving the opportunity for more precise targeting of antimycotic therapy.

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.

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.