Bloodstream Infections Caused by Magnusiomyces capitatus and Magnusiomyces clavatus: Epidemiological, Clinical, and Microbiological Features of Two Emerging Yeast Species.

Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. Our objectives were to determine the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001 to 2020. In seven institutions, a total of 34 Magnusiomyces BSI were identified. Identification was done by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Of the 34 isolates, M. clavatus was more common (n = 24) than M. capitatus (n = 10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with hemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus/M. capitatus was observed for voriconazole (MIC50, 0.03/0.125 mg/L), followed by posaconazole (MIC50, 0.125/0.25 mg/L). M. clavatus isolates showed overall lower MICs than M. capitatus. With the exception of amphotericin B, low essential agreement between gradient test and microdilution was recorded for all antifungals (0 to 70%). Both species showed distinct morphologic traits on ChromAgar Orientation medium and Columbia blood agar, which can be used for differentiation if no MALDI-TOF MS or molecular identification is available. In conclusion, most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

ß-1,3-d-Glucan and Galactomannan as Biomarkers for the Detection of Invasive Geotrichum and Magnusiomyces Infections: a Retrospective Evaluation.

Magnusiomyces and Geotrichum species are ascomycetous yeasts that can cause potentially life-threatening invasive fungal infections commonly referred to as geotrichosis. In this study, we aimed to estimate the incidence and mortality of these infections in a German tertiary care center. Furthermore, we evaluated the suitability of the fungal biomarkers galactomannan (GM) and ß-1,3-d-glucan (BDG), which are both recommended as surrogate markers for Magnusiomyces capitatus infection by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the European Confederation of Medical Mycology (ECMM) joint clinical guidelines for the diagnosis and management of rare invasive yeast infections for detection of invasive geotrichosis. Cases meeting the inclusion criteria for invasive Magnusiomyces/Geotrichum infection were retrospectively identified. Serum samples and culture supernatants were analyzed with two commercially available fungal antigen tests (Platelia Aspergillus Ag EIA and Wako ß-glucan test). For a control cohort, outpatient samples sent for lues testing were included. Thirty-eight cases of Magnusiomyces/Geotrichum infection were identified over an 11-year observation period. In the majority of cases, the fungus was isolated from intra-abdominal specimens of patients with a history of abdominal surgery/procedures (n = 32). All cases of fungemia occurred exclusively in haemato-oncologic patients (n = 14). Thirty-day survival was 42% in the fungemia and 43% in the intra-abdominal geotrichosis group. Serum samples were available for 23 patients (14 bloodstream and nine intra-abdominal infections). While BDG sensitivity was 65%, none of the sera was GM positive. This finding was supported by in vitro experiments analyzing fungal culture supernatants: M. capitatus secretes significant amounts of BDG but not GM. Specificity was 96% for BDG and 100% for GM. Magnusiomyces and Geotrichum infections are not limited to haemato-oncologic patients. Contrasting the current ESCMID/ECMM recommendation, our results indicate that GM is no suitable biomarker for the diagnosis of Magnusiomyces infection. Contrarily, BDG sensitivity is comparable to that of candidemia.

Temperate phages promote colicin-dependent fitness of Salmonella enterica serovar Typhimurium.

Bacteria employ bacteriocins for interference competition in microbial ecosystems. Colicin Ib (ColIb), a pore-forming bacteriocin, confers a significant fitness benefit to Salmonella enterica serovar Typhimurium (S. Tm) in competition against commensal Escherichia coli in the gut. ColIb is released from S. Tm into the environment, where it kills susceptible competitors. However, colicin-specific release proteins, as they are known for other colicins, have not been identified in case of ColIb. Thus, its release mechanism has remained unclear. In the current study, we have established a new link between ColIb release and lysis activity of temperate, lambdoid phages. By the use of phage-cured S. Tm mutant strains, we show that the presence of temperate phages and their lysis genes is necessary and sufficient for release of active ColIb into the culture supernatant. Furthermore, phage-mediated lysis significantly enhanced S. Tm fitness in competition against a ColIb-susceptible competitor. Finally, transduction with the lambdoid phage 933W rescued the defect of E. coli strain MG1655 with respect to ColIb release. In conclusion, ColIb is released from bacteria in the course of phage lysis. Our data reveal a new mechanism for colicin release and point out a novel function of temperate phages in enhancing colicin-dependent bacterial fitness.

Inflammation fuels colicin Ib-dependent competition of Salmonella serovar Typhimurium and E. coli in enterobacterial blooms.

The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae ("inflammation-inflicted blooms"). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family.

Comparison of the duplex-destabilizing effects of nucleobase-caged oligonucleotides.

Nucleobase-caged oligonucleotide residues have photolabile "caging groups" that prevent the formation of Watson-Crick base pairs until the unmodified nucleobase is restored in a photolysis event. This principle can be used to put a growing variety of powerful nucleic acid-based applications under the precise spatiotemporal control using light as an addressing mechanism. Examples for applications include light control of transcription, RNAi, nucleic acid folding, primer extension, and restriction endonuclease as well as DNAzyme, aptamer, and antisense activity. However, a comparison of the duplex-destabilization properties of the various caged residues that have been used up to date and rules for achieving a maximal duplex destabilization with a minimum amount of modified residues are still missing. We present both a comparison of the duplex-destabilizing capabilities of various nucleobase-caged residues and address the question of influence on neighboring base pairs.

Evaluation of a Novel CLIA Monotest Assay for the Detection of Anti-Hepatitis E Virus-IgG and IgM: A Retrospective Comparison with a Line Blot and an ELISA.

Despite the increasing relevance of Hepatitis E, an emerging disease endemic in developing and with increasing numbers of sporadic cases in industrialized countries, commercial tests are mainly based on batch oriented serological assays. In this retrospective study, we compared a line immunoassay (LIA; recomLine HEV, Mikrogen) and an ELISA (EIA; Anti-Hepatitis E Virus ELISA, Euroimmun) with a novel chemoluminescence immunoassay in a monotest format (CLIA; Hepatitis E VirClia, Vircell). Twenty sera of PCR proven cases of hepatitis E and 68 blood samples serologically pre-characterized were included. Applying the WHO reference standard, the CLIA demonstrated the highest analytical sensitivity for IgG and IgM. The combinations of CLIA/EIA (IgG and IgM) and CLIA/LIA (IgG) measurements showed substantial correlation. Compared to overall antibody detection (seropositivity in ≥2 assays), CLIA correlation was excellent, outperforming LIA (IgM) and EIA (IgG and IgM). Minor IgM cross reactivity in samples of patients with acute EBV infection was observed in all three assays. The CLIA showed good performance in diagnostic samples compared to established LIA and EIA assays. Due to its ready-to-use monotest format, the CLIA allows simple, time- and cost-effective handling of single samples. These qualities make the assay suitable for diagnostics, especially in the emergency setting and for low-throughput laboratories.

Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens.

Stx2 is the major virulence factor of EHEC and is associated with an increased risk for HUS in infected patients. The conditions influencing its expression in the intestinal tract are largely unknown. For optimal management and treatment of infected patients, the identification of environmental conditions modulating Stx2 levels in the human gut is of central importance. In this study, we established a set of chromosomal stx2 reporter assays. One system is based on superfolder GFP (sfGFP) using a T7 polymerase/T7 promoter-based amplification loop. This reporter can be used to analyze stx2 expression at the single-cell level using FACSs and fluorescence microscopy. The other system is based on the cytosolic release of the Gaussia princeps luciferase (gluc). This latter reporter proves to be a highly sensitive and scalable reporter assay that can be used to quantify reporter protein in the culture supernatant. We envision that this new set of reporter tools will be highly useful to comprehensively analyze the influence of environmental and host factors, including drugs, small metabolites and the microbiota, on Stx2 release and thereby serve the identification of risk factors and new therapies in Stx-mediated pathologies.

DNA minicircles connected via G-quadruplex interaction modules.

G-quadruplexes are becoming reliable alternative interaction modules for the construction of DNA nanoarchitectures due to their prompt inducibility by salts. In this Full Paper, we report the design and synthesis of two different DNA minicircles equipped with G-rich appendixes that can self-hybridize into a G-quadruplex, which acts as a DNA recruiter and glue. Both minicircles, one containing a hairpin-like G-rich region and the other an open tuning-fork-like G-rich region, have the potential to form DNA G-nanoconstructs but only the tuning-fork minicircle does so. Incubation of the tuning-fork minicircle with Na(+) and Ni(2+) results in the formation of minicircle dimers, while K(+) and Sr(2+) unexpectedly induce the formation of multimers. Moreover, a catenated DNA nanoconstruct is obtained when the components of the hairpin minicircle are incubated with K(+) or Na(+) and assembled in a stepwise sequence. All nanoconstructs are visualized by atomic force microscopy.

Usability of rectal swabs for microbiome sampling in a cohort study of hematological and oncological patients.

OBJECTIVES: Large-scale clinical studies investigating associations between intestinal microbiota signatures and human diseases usually rely on stool samples. However, the timing of repeated stool sample collection cannot be predefined in longitudinal settings. Rectal swabs, being straightforward to obtain, have the potential to overcome this drawback. Therefore, we assessed the usability of rectal swabs for microbiome sampling in a cohort of hematological and oncological patients. STUDY DESIGN: We used a pipeline for intestinal microbiota analysis from deep rectal swabs which was established and validated with test samples and negative controls. Consecutively, a cohort of patients from hematology and oncology wards was established and weekly deep rectal swabs taken during their admissions and re-admissions. RESULTS: Validation of our newly developed pipeline for intestinal microbiota analysis from rectal swabs revealed consistent and reproducible results. Over a period of nine months, 418 rectal swabs were collected longitudinally from 41 patients. Adherence to the intended sampling protocol was 97%. After DNA extraction, sequencing, read pre-processing and filtering of chimeric sequences, 405 of 418 samples (96.9%) were eligible for further analyses. Follow-up samples and those taken under current antibiotic exposure showed a significant decrease in alpha diversity as compared to baseline samples. Microbial domination occurred most frequently by Enterococcaceae (99 samples, 24.4%) on family level and Enterococcus (90 samples, 22.2%) on genus level. Furthermore, we noticed a high abundance of potential skin commensals in 99 samples (24.4%). SUMMARY: Deep rectal swabs were shown to be reliable for microbiome sampling and analysis, with practical advantages related to high sampling adherence, easy timing, transport and storage. The relatively high abundance of putative skin commensals in this patient cohort may be of potential interest and should be further investigated. Generally, previous findings on alpha diversity dynamics obtained from stool samples were confirmed.

Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium.

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a "common good" for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib), a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single- and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP+ S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP+ and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut.