Integrated molecular, phenotypic and epidemiological surveillance of antimicrobial resistance in Neisseria gonorrhoeae in Germany.

Numbers of infections with Neisseria gonorrhoeae are among the top three sexually transmitted infections (STI) worldwide. In addition, the emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae pose an important public-health issue. The integration of genomic, phenotypic and epidemiological data to monitor Neisseria gonorrhoeae fosters our understanding of the emergence and spread of AMR in Neisseria gonorrhoeae and helps to inform therapy guidelines and intervention strategies. Thus, the Gonococcal resistance surveillance (Go-Surv-AMR) was implemented at the Robert Koch Institute in Germany in 2021 to obtain molecular, phenotypic and epidemiological data on Neisseria gonorrhoeae isolated in Germany. Here, we describe the structure and aims of Go-Surv-AMR. Furthermore, we point out future directions of Go-Surv-AMR to improve the integrated genomic surveillance of Neisseria gonorrhoeae. In this context we discuss current and prospective sequencing approaches and the information derived from their application. Moreover, we highlight the importance of combining phenotypic and WGS data to monitor the evolution of AMR in Neisseria gonorrhoeae in Germany. The implementation and constant development of techniques and tools to improve the genomic surveillance of Neisseria gonorrhoeae will be important in coming years.

A shift from travel-associated cases to autochthonous transmission with Berlin as epicentre of the monkeypox outbreak in Germany, May to June 2022.

By 22 June 2022, 521 cases of monkeypox were notified in Germany. The median age was 38 years (IQR: 32-44); all cases were men. In Berlin, where 69% of all cases occurred, almost all were men who have sex with men. Monkeypox virus likely circulated unrecognised in Berlin before early May. Since mid-May, we observed a shift from travel-associated infections to mainly autochthonous transmission that predominantly took place in Berlin, often in association with visits to clubs and parties.

Characterization of Methicillin-resistant Staphylococcus aureus From Children at Hospital Admission: Experiences From a Hospital in a German Metropolitan Area.

BACKGROUND: Since the 1990s, community-associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) are described as emerging independent of health care. CA-MRSA is associated with the colonization and infection of healthy, immunocompetent younger individuals. While skin and soft tissue infections (SSTI) are predominant, life-threatening syndromes can also occur. METHODS: In this retrospective study, we investigated MRSA stains isolated from community-onset infections and from MRSA screening of children at admission to a tertiary-care hospital in 2012-2018. In total, 102 isolates were subjected to antibiotic susceptibility testing by broth microdilution, spa -typing, multilocus sequence typing, SCC mec typing and virulence/resistance gene detection by polymerase chain reaction. RESULTS: The majority of isolates originated from community-onset infections (80/102), of these primarily from SSTI (70/80). Additional strains were isolated by MRSA screening (22/102). In total 61.8% of the MRSA carried the gene for the Panton-Valentine leukocidin ( lukPV ). Molecular characterization of isolates revealed various epidemic MRSA clones, circulating in both community and hospital settings. Most prevalent epidemic lineages were isolates of the "European CA-MRSA clone" (CC80-MRSA-IV), the "Bengal Bay clone" (ST772-MRSA-V), or the "USA300 NAE clone" (ST8-MRSA-IVa). CONCLUSIONS: Our data highlight the importance of CA-MRSA causing SSTI in children. More frequent microbiological and molecular analysis of these strains is important for targeted treatment and can provide valuable data for molecular surveillance of the pathogen.

Markedly decreasing azithromycin susceptibility of Neisseria gonorrhoeae, Germany, 2014 to 2021.

We monitored antimicrobial susceptibility developments of Neisseria gonorrhoeae in Germany from January 2014 to May 2021. The proportion of isolates with azithromycin minimum inhibitory concentrations above the epidemiological cut-off increased substantially, from 1.3% in 2014 to 12.2% in 2020. Preliminary data from 2021 showed a further rise (January to May: 20.7%). Therefore, azithromycin as part of the recommended dual therapy in Germany for non-adherent patients is challenged. Antimicrobial susceptibility testing in clinical practice is crucial and continuous susceptibility surveillance indispensable.

The Mosaic mtr Locus as Major Genetic Determinant of Azithromycin Resistance of Neisseria gonorrhoeae-Germany, 2018.

Within the German Gonococcal Resistance Network's (GORENET) Neisseria gonorrhoeae (NG) sample collection, azithromycin-resistant NG isolates increased from 4.3% in 2016 to 9.2% in 2018. We aim to understand this observed increase using whole genome sequencing of NG isolates combined with epidemiological and clinical data. Reduced susceptibility to azithromycin in 2018 was predominately clonal (NG multiantigen sequence typing G12302) and could mainly be attributed to the recently described mosaic-like mtr locus. Our data suggest that, together with horizontal gene transfer of resistance determinants and well-established point mutations, international spread of resistant lineages plays a major role regarding azithromycin resistance in Germany.

External quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in primary laboratories in Germany.

BACKGROUND: Worldwide, an increase in antimicrobial resistance (AMR) of Neisseria gonorrhoeae has been observed. Until now, no protocol for an external quality assessment (EQA) has been available for Germany. The German gonococcal resistance network (GORENET) performed an EQA of primary laboratories in Germany in order to assess quality of antibiotic susceptibility testing, to gain information about laboratory procedures and to assess the impact of these procedures on test results. METHODS: Laboratories assessed drug susceptibility to cefixime, ceftriaxone, azithromycin, penicillin and ciprofloxacin for five N. gonorrhoeae strains, using their standard laboratory protocols. Minimal inhibitory concentrations (MICs) were compared to World Health Organisation (WHO) consensus results (or, if not available, reference laboratory results), while deviation by +/- one doubling dilution was accepted. Data on laboratory procedures were collected via a standardised questionnaire. Generalized linear models and conditional inference trees (CTREE) were used to assess relationships between laboratory procedures and testing outcomes. RESULTS: Twenty-one primary laboratories participated in the EQA in June 2018. 96% of ciprofloxacin MICs were reported within accepted deviations, as well as 88% for cefixime, 85% for ceftriaxone, 79% for penicillin and 70% for azithromycin. The use of interpretation standards and general laboratory procedures like agar base, incubation settings or the use of control strains strongly differed between laboratories. In statistical analysis, incubation time of cultures < 24 h was associated with correct measurements. Additionally, a 5% CO2 concentration was associated with correct results regarding azithromycin compared to 3%. CTREE analysis showed that incubation time, humidity and CO2 concentration had the greatest influence on the average deviation from consensus results. CONCLUSIONS: In conclusion, we report the development of a protocol for N. gonorrhoeae antimicrobial susceptibility testing in Germany. While testing results were in accordance with the expected consensus results in 70-96%, depending on the antibiotic agent, laboratory methodology was heterogeneous and may significantly affect the testing quality. We therefore recommend the development of a standard operating procedure (SOP) for N. gonorrhoeae susceptibility testing in Germany.

Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017.

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients' samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.

Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

Betamethasone prevents human rhinovirus- and cigarette smoke- induced loss of respiratory epithelial barrier function.

The respiratory epithelium is a barrier against pathogens and allergens and a target for therapy in respiratory allergy, asthma and chronic obstructive pulmonary disease (COPD). We investigated barrier-damaging factors and protective factors by real-time measurement of respiratory cell barrier integrity. Barrier integrity to cigarette smoke extract (CSE), house dust mite (HDM) extract, interferon-γ (IFN-γ) or human rhinovirus (HRV) infection alone or in combination was assessed. Corticosteroids, lipopolysaccharide (LPS), and nasal mucus proteins were tested for their ability to prevent loss of barrier integrity. Real-time impedance-based measurement revealed different patterns of CSE-, HDM-, IFN-γ- and HRV-induced damage. When per se non-damaging concentrations of harmful factors were combined, a synergetic effect was observed only for CSE and HDM. Betamethasone prevented the damaging effect of HRV and CSE, but not damage caused by HDM or IFN-γ. Real-time impedance-based measurement of respiratory epithelial barrier function is useful to study factors, which are harmful or protective. The identification of a synergetic damaging effect of CSE and HDM as well as the finding that Betamethasone protects against HRV- and CSE-induced damage may be important for asthma and COPD.

Extracorporeal IgE Immunoadsorption in Allergic Asthma: Safety and Efficacy.

BACKGROUND: Prevention of IgE-binding to cellular IgE-receptors by anti-IgE (Omalizumab) is clinically effective in allergic asthma, but limited by IgE threshold-levels. To overcome this limitation, we developed a single-use IgE immunoadsorber column (IgEnio). IgEnio is based on a recombinant, IgE-specific antibody fragment and can be used for the specific extracorporeal desorption of IgE. OBJECTIVE: To study safety and efficacy of IgEnio regarding the selective depletion of IgE in a randomized, open-label, controlled pilot trial in patients with allergic asthma and to investigate if IgEnio can bind IgE-Omalizumab immune complexes. METHODS: Fifteen subjects were enrolled and randomly assigned to the treatment group (n=10) or to the control group (n=5). Immunoadsorption was done by veno-venous approach, processing the twofold calculated plasma volume during each treatment. A minimum average IgE-depletion of 50% after the last cycle in the intention-to-treat population was defined as primary endpoint. Safety of the treatment was studied as secondary endpoint. In addition, possible changes in allergen-specific sensitivity were investigated, as well as clinical effects by peak flow measurement and symptom-recording. The depletion of IgE-Omalizumab immune complexes was studied in vitro. The study was registered at clinicaltrials.gov (NCT02096237) and conducted from December 2013 to July 2014. RESULTS: IgE immunoadsorption with IgEnio selectively depleted 86.2% (±5.1% SD) of IgE until the end of the last cycle (p<0.0001). Removal of pollen allergen-specific IgE was associated with a reduction of allergen-specific basophil-sensitivity and prevented increases of allergen-specific skin-sensitivity and clinical symptoms during pollen seasons. IgEnio also depleted IgE-Omalizumab immune complexes in vitro. The therapy under investigation was safe and well-tolerated. During a total of 81 aphereses, 2 severe adverse events (SAE) were recorded, one of which, an episode of acute dyspnea, possibly was related to the treatment and resolved after administration of antihistamines and corticosteroids. CONCLUSIONS: This pilot study indicates that IgE immunoadsorption with IgEnio may be used to treat patients with pollen-induced allergic asthma. Furthermore, the treatment could render allergic patients with highly elevated IgE-levels eligible for the administration of Omalizumab and facilitate the desorption of IgE-Omalizumab complexes. This study was funded by Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany.

The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1.

In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadiiIMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells.

BACKGROUND: Increasing evidence suggests that the low-affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen-specific T cells through IgE-facilitated allergen presentation. OBJECTIVE: We sought to determine the number of CD23 molecules on immune cells in allergic patients and to investigate whether the number of CD23 molecules on antigen-presenting cells is associated with IgE levels and influences allergen uptake and allergen-specific T-cell activation. METHODS: Numbers of CD23 molecules on immune cells of allergic patients were quantified by using flow cytometry with QuantiBRITE beads and compared with total and allergen-specific IgE levels, as well as with allergen-induced immediate skin reactivity. Allergen uptake and allergen-specific T-cell activation in relation to CD23 surface density were determined by using flow cytometry in combination with confocal microscopy and T cells transfected with the T-cell receptor specific for the birch pollen allergen Bet v 1, respectively. Defined IgE-allergen immune complexes were formed with human monoclonal allergen-specific IgE and Bet v 1. RESULTS: In allergic patients the vast majority of CD23 molecules were expressed on naive IgD+ B cells. The density of CD23 molecules on B cells but not the number of CD23+ cells correlated with total IgE levels (RS = 0.53, P = .03) and allergen-induced skin reactions (RS = 0.63, P = .008). Uptake of allergen-IgE complexes into B cells and activation of allergen-specific T cells depended on IgE binding to CD23 and were associated with CD23 surface density. Addition of monoclonal IgE to cultured PBMCs significantly (P = .04) increased CD23 expression on B cells. CONCLUSION: CD23 surface density on B cells of allergic patients is correlated with allergen-specific IgE levels and determines allergen uptake and subsequent activation of T cells.

Critical and direct involvement of the CD23 stalk region in IgE binding.

BACKGROUND: The low-affinity receptor for IgE, FcεRII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of transepithelial allergen migration. OBJECTIVE: We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. METHODS: We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N-linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. RESULTS: A hitherto unknown IgE-binding site was mapped on the stalk region of CD23, and the non-N-glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti-IgE antibody, omalizumab, which inhibits IgE binding to FcεRI, also inhibited IgE binding to CD23. CONCLUSION: Our results provide a new model for the CD23-IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti-IgE antibody omalizumab.

Guidance on allergenicity assessment of genetically modified plants.

This document provides supplementary guidance on specific topics for the allergenicity risk assessment of genetically modified plants. In particular, it supplements general recommendations outlined in previous EFSA GMO Panel guidelines and Implementing Regulation (EU) No 503/2013. The topics addressed are non-IgE-mediated adverse immune reactions to foods, in vitro protein digestibility tests and endogenous allergenicity. New scientific and regulatory developments regarding these three topics are described in this document. Considerations on the practical implementation of those developments in the risk assessment of genetically modified plants are discussed and recommended, where appropriate.

Genomic manipulations in alkaliphilic haloarchaea demonstrated by a gene disruption in Natrialba magadii.

Alkaliphilic haloarchaea, a distinct physiological group from the closely related neutrophilic haloarchaea, represent an underutilized resource for basic research and industrial applications. In contrast to the neutrophilic haloarchaea, no reports on genomic manipulations in haloalkaliphiles have been published until now. Genomic manipulations via homologous recombination are useful for basic research. In this study, we demonstrate the possibility for this strategy in alkaliphilic haloarchaea for the first time. In a previous study, we developed a PEG-mediated transformation technique for alkaliphilic haloarchaea that was deployed in this study to deliver a gene disruption cassette into the model organism Natrialba magadii. The gene encoding for the well-studied Natrialba extracellular protease was successfully disrupted by a recombination marker gene, demonstrating a proof of principle for the usability of homologous recombination for genomic manipulations in alkaliphilic haloarchaea. Since halo(alkali)philic Archaea are polyploid, a selection process was applied in order to obtain a mutant strain containing exclusively disrupted genes. The resulting strain exhibited no proteolytic activity measurable by an azo-casein assay. Complementation was able to restore proteolytic activity. The expression pattern of the Natrialba extracellular protease was different in the complemented strain.

Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential IgE epitopes indicates that sensitization to alpha-purothionin occurs via the gut. Both allergens can be used for in-vitro diagnosis of wheat food allergy. The induction of blocking IgG antibodies suggests the usefulness for immunotherapy.