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Arcobacter (A.) butzleri, the most widespread species within the genus Arcobacter, is considered as an emerging pathogen causing gastroenteritis in humans. Here, we performed a comparative genome-wide analysis of 40 A. butzleri strains from Lithuania to determine the genetic relationship, pangenome structure, putative virulence, and potential antimicrobial- and heavy-metal-resistance genes. Core genome single nucleotide polymorphism (cgSNP) analysis revealed low within-group variability (≤4 SNPs) between three milk strains (RCM42, RCM65, RCM80) and one human strain (H19). Regardless of the type of input (i.e., cgSNPs, accessory genome, virulome, resistome), these strains showed a recurrent phylogenetic and hierarchical grouping pattern. A. butzleri demonstrated a relatively large and highly variable accessory genome (comprising of 6284 genes with around 50% of them identified as singletons) that only partially correlated to the isolation source. Downstream analysis of the genomes resulted in the detection of 115 putative antimicrobial- and heavy-metal-resistance genes and 136 potential virulence factors that are associated with the induction of infection in host (e.g., cadF, degP, iamA), survival and environmental adaptation (e.g., flagellar genes, CheA-CheY chemotaxis system, urease cluster). This study provides additional knowledge for a better A. butzleri-related risk assessment and highlights the need for further genomic epidemiology studies in Lithuania and other countries.

In the hyper-arid Atacama Desert, microbial life thrives near its “dry limit” in scarcely distributed habitats. Fracture networks of salt-poor sand wedges outlining salt-cemented polygons on alluvial surfaces in the Yungay region (Chile) represent potential microbial habitats. The degree of soil habitability at the surface (0–5 cm depth) and subsurface (10–15 cm depth) of a polygon and adjacent sand wedge was assessed before and up to 42 days after a 20 mm simulated rain experiment through the abundance of phospholipid fatty acids (PLFAs). Mineralogical composition, salinity, pH, electrical and thermal conductivity, water content, and water activity were analyzed for their relevance to habitability. After wetting, the PLFA content exclusively increased steadily with time in the polygon subsurface indicating the growth of an indigenous bacterial community. This increased habitability is presumably related to the soil's ability to retain water for at least 6 weeks at this depth. The lack of a continuous growth signal at the surface is likely due to rapid desiccation. In the sand wedge subsurface, the increase in PLFA content is not continuous despite the water activity being >0.9. The reason for this remains unclear but indicates that not only water availability is relevant for habitability but also the here described soil heterogeneities might impact the detection of the microbial response. Yet, the increasing PLFA trend in the polygon subsurface emphasized its relevance as a saline microbial habitat in an otherwise hostile environment, which could have implications for the assessment of soil habitability on Mars.

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development.

Background The biofilm formation of nontuberculous mycobacteria (NTM) gained more and more attention in the past years. NTM biofilms were found in contaminated medical devices causing globel outbreaks and in the lung cavities of patients where they complicate treatment. These findings raised the attention on NTM biofilms. Methods In the present study, a porous glass bead (PGB) biofilm cultivation model was adjusted to Mycobac- terium abescessus and Mycobacterium chimaera. The biofilms were cultivated in different media and the bacteria quantified using multiple experimental approaches (CFU, ATP and qPCR quantification). The biofilms were further analysed in structure and composition and their susceptibility towards dis- infectants and antibiotics was tested. Further, the proteome and transcriptome expression profiles of two M. chimaera isolates were analysed to identify biofilm specific genes. Results All tested isolates formed surface attached biofilms, including rough M. abscessus isolates. Strong dif- ferences in the structures were found between the isolates also within one species and, in M. abscessus, even between different colony morphotypes of the same isolate. Moreover, it was found that the used medium had a fundamental influence on growth and structure of the cultivated biofilms, affecting for example the expression of a cording phenotype in smooth M. abscessus isolates. In all cases the biofilms showed an enhanced biomass of extracellular matrix (ECM) components when compared to the corresponding suspension. Most impressively it was found that the biofilm of the outbreak strain ZUERICH-1 consists of an extremely enhanced amount of protein compared to the other M. chimaera isolates. The differences in the composition of the biofilms were less isolate specific in M. abscessus than in M. chimaera. The proteome and the transcriptome analysis revealed the expression of biofilm specific genes (e.g. glmM, glgX, pepN ) including genes of the type VII secretion systems. Finally, it was found that that the biofilms provide enhanced tolerance to antibiotics and disinfectants with the exception of sodium hypochlorite. In this case, the suspension of all M. chimaera isolates were more tolerant than the corresponding biofilm. An interesting finding was, that the outbreak strain M. chimaera ZUERICH-1 presents an enhanced tolerance to peracetic acid (PAA) in compari- son to the other isolates. It is important to mention, that commonly recommended concentrations of disinfectants were not able eradicate the biofilms of several isolates tested. Conclusion The PGB model is well suited for cultivation of different NTM biofilms and is highly applicable to various analysis methods. The biofilms of the tested species provide very different characteristics highly depending on the cultivation condititons. It was possible to identify biofilm specific genes, which could be a first step to define a basic NTM biofilm genotype. Finally it was found that antimicrobials commonly used to inactivate NTM fail to eradicate or reduce the biofilms. It will be necessary to adjust the common laboratory methods to include NTM biofilms.

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced A. hydrophila strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.

Under a changing climate, soil fungal communities will increasingly be subject to periods of heat stress. These periods can affect the performance of individual fungi and their competition for space and resources. Competition between fungi is strongly controlled by the exudation of inhibitory compounds, resulting in different competitive outcomes that range from overgrowth of the inferior competitor to a deadlock, where the competing fungi inhibit each other. As heat stress can alter the competitive outcome between fungi, the community composition can also change strongly. So far, a general understanding of the mechanisms that drive the competitive outcome between fungi under heat stress is still missing. However, this understanding is essential to assess important community functions, such as decomposition or mediation of plant nutrition, which strongly depend on the fungal community composition. Here, we used a partial differential equation (PDE) model simulating two fungal competitors in a two-dimensional space, to mechanistically explain the observed change of fungal competition under heat stress. The model describes mycelial growth, the production and secretion of antifungal compounds and the synthesis of heat shock proteins of interacting colonies. We found a heat stress-induced lag phase favouring the accumulation of antifungal compounds and the build-up of inhibitor fields. This led to a qualitative change of the competitive outcome, reducing the occurrence of overgrowth by two thirds. The changes in competitive outcome favoured slower growing species, which benefit more strongly from the additional time during a stress-induced lag to build up a defence or block territory that would otherwise be quickly claimed by faster competitors. Our work is an important step towards understanding how environmental changes may lead to qualitative changes in competitive outcomes. Our results show the importance of explicitly including species interactions into studies of climate change effects.

Arbuscular mycorrhizal (AM) fungi have gained immense research interest due to their symbiotic relation with roots of most terrestrial land plants. Diverse positive effects on plant health and fitness, as well as a high ecological relevance for many other below-and aboveground processes have been recognized. Despite growing research ambitions, sufficient knowledge on drivers and their relative impacts on AM fungal communities at varying scales and ecosystems is lacking. Very little attention has been paid to the interrelation of the spatial structure of AM host plants (here meaning the small- scale distribution of host plant individuals or populations) and AM fungal community dynamics, although deeper insights could move forward the development of theoretical community frameworks applicable to these important endosymbionts. The work presented in this thesis aimed to study the effects of host plant spatial structure for the establishment of mycorrhizal symbiosis as well as for AM fungal diversity and community composition. To do so, controlled experiments mimicking micro- landscapes of AM fungal habitats were conducted and compared with observational data from temperate forests in Germany. In the experiments, we used meta-community designs to test the responses of root colonization, AM fungal diversity and community composition to levels of habitat connectance or habitat heterogeneity. We assayed rates of root colonization across forests sites differing in AM-associating plant cover. For assessing the importance of further spatio-temporal parameters and host-specificity we surveyed AM fungal communities within one of the forest sites over two years. As expected, we observed a consistent pattern of higher rates of root colonization at a higher proximity of host plants, which in the case of the field study was a higher cover of AM- associating woody plants and in the case of the experiment treatments a higher connectance of host plant patches. There were generally no clear effects of habitat structure on AM fungal diversity and community composition in both the field and the experimental study. In the field, the main finding was that spatial parameters, followed by host identity, more strongly affected AM fungal assemblages than temporal parameters. In the experiments, we observed within-experimental-unit differences of beta-diversity between pairs of long- and short-distance patches as well as between pairs of different habitat quality, which rendered those communities less predictable. In conclusion, we present empirical evidence for a co-variation of AM plant host structure and AM fungal abundance. These findings are of importance for the restoration or management of natural forest habitats, as well as for sustainable agriculture. Furthermore, this work supports previous findings of high stochasticity in AM fungal communities, highlighting the need for further research into spatial scale-specific drivers of this important organism group.

E. faecium SF68 is a licensed probiotic, which has been shown to ameliorate symptoms of inflammation in enteritis and to reduce the incidence and severity of diarrhea in humans and animals. In vivo trials with weaning piglets performed in our laboratory or in cooperation with our laboratory, yielded controversial observations on the anti-inflammatory effects of E. faecium SF68. Supplementation with the probiotic in animals was reported to contribute to reduced expression of immune-associated genes in intestinal tissues and associated lymphoid organs. Moreover, these animals had lower amounts of serum IgG and fecal IgA, and reduced CD8+ intraepithelial lymphocyte populations. Piglets supplemented with E. faecium SF68 showed higher colonization and greater pathogen shedding in Salmonella infection studies. Since the alterations observed were mainly associated with local innate immune responses, this doctoral study focused mainly on the direct immunomodulatory effects of E. faecium SF68 on intestinal epithelial cells in an in vitro model. NF-κB, one of the key initiators of innate immune responses in intestinal epithelial cells encountering microbiota and invasive enteric pathogens, was selected as a reporter for determination of the effects of E. faecium SF68 on immune response modulation in intestinal epithelial cells. Cell-free, whole bacterial lysates of E. faecium SF68 inhibited NF-κB activation in intestinal epithelial cells from different host species. This inhibitory effect occurred in a reversible manner; after removal of the lysate and addition of fresh culture medium, cells recovered their NF-κB activity. Cells treated with the bacterial lysates ceased to proliferate, based on the results of host cell cycle protein Ki67 staining. Porcine intestinal epithelial cells treated with the probiotic lysate contained lower levels of phosphorylated p65 subunit of NF-κB at serine 536 compared to untreated cells. Phosphorylation at this amino acid is one of the most studied modifications involved in the transcriptional activity of p65 subunit. Moreover, fluorescence microscopy images of porcine intestinal cells suggested a dysregulation in the trafficking of NF-κB between the cytoplasm and nucleus. Ammonium sulfate (AS) fractionation of proteins of the E. faecium SF68 lysates and analysis of the proteins present in different AS-fractions revealed that several enzymes involved in arginine metabolism are present in the active AS-fraction. Other commensal and clinical E. faecium isolates, as well as isolates of other enterococcal species possessing enzymes of arginine deiminase pathway including E. faecalis, E. hirae and E. durans, exhibited the same inhibitory effect on NF-κB activity of IPEC-J2 cells. E. gallinarum and E. casseliflavus species were the two exceptions. These two species are the only motile enterococcal species expressing flagellin which is a strong stimulant of inflammatory pathways such as NF-κB, particularly in our in vitro cell model. Experiments examining pre- and co-incubation of host cells with E. faecium SF68 lysates and different Toll-like receptor (TLR)- and NOD-like receptor (NLR)-ligands showed that cells pre-treated with the E. faecium SF68 bacterial lysates were severely impaired in their NF-κB activation responses to these pathogen-associated molecular pattern (PAMP) signals. Lysates from strains of other enterococcal species with no AD pathway activity (E. avium, E. cecorum and E. raffinosus) did not inhibit the NF-κB activity of treated IPEC-J2 cells. The arcA gene of E. faecium SF68 encoding arginine deiminase was cloned into an E. coli-Enterococcus shuttle vector and introduced into an E. avium strain which showed no inhibitory effects on NF-κB activity. Transformants of E. avium constitutively expressing arginine deiminase of E. faecium SF68 showed the same NF-κB-inhibitory effects on porcine and human intestinal epithelial cell lines as E. faecium SF68. Comparison of results with the Streptococcus suis 10 strain, another bacterial genus possessing AD-pathway enzymes, exhibited the same results. Furthermore, an arcA knockout mutant of S. suis 10 no longer showed inhibition of NF-κB activity of host cells and purified recombinant arginine deiminase of S. suis 10 was shown to have the same inhibitory effects on NF-κB activity as the cell-free, whole bacterial lysates of S. suis 10 and E. faecium SF68. Finally, arginine supplementation of the cell culture medium eliminated the inhibitory effects on NF-κB activity mediated by arginine deiminase or bacterial lysates containing arginine deiminase. In summary, we postulate that the reported anti-inflammatory effects of E. faecium SF68 in vitro and likely in vivo, are associated with arginine deiminase-mediated arginine depletion. Beneficial bacteria in general can have a large number of molecules and metabolites, allowing them to interact with intestinal epithelial and immune cells. Therefore, the immunomodulatory effects elicited by beneficial microbes in the gut may not be limited to a single factor. Additionally, the health condition of the recipients, their age and immune status can determine the outcome of beneficial microbe administration. For instance, neonates with underdeveloped immune systems, not fully shaped microbiota and higher metabolic activity are more sensitive to arginine depletion (Flynn et al. 2002). Moreover, the concurrent presence of an intestinal pathogen such as Salmonella or Giardia, which use arginine depletion as a defensive strategy to hinder the host iNOS production and proliferation of immune cells, can aggravate the clinical condition. These pathogens promote arginine depletion by their own AD-pathway enzymes or induction of arginine consuming enzymes in the host cells. The mechanisms of action of beneficial microbes should be studied well and in detail. More importantly, the assessments and decisions on the administration of a beneficial microbe as therapeutic or preventive measure should be made cautiously and individually for each case to optimize the use of these advantageous biological tools., E. faecium SF68 ist ein lizenziertes Probiotikum, dass nachweislich Entzündungssymptome bei Enteritis lindert und die Häufigkeit und Schwere von Durchfall bei Menschen und Tier reduziert. In einigen In vivo-Studien, die in unserem Labor oder in Zusammenarbeit mit unserem Labor durchgeführt wurden, wurden kontroverse Beobachtungen über entzündungshemmende Effekte von E. faecium SF68 bei Ferkeln gemacht. Es wurde berichtet, dass die Supplementierung von Tieren mit dem Probiotikum zu einer geringeren Expression von immun-assoziierten Genen des Darmgewebes und der assoziierten lymphoiden Organe beiträgt. Außerdem hatten diese Tiere ein niedrigeres Serum-IgG und fäkales IgA. Darüber hinaus wurden bei diesen Tieren geringere CD8+ intraepitheliale Lymphozyten-populationen nachgewiesen. Ferkel, die gleichzeitig mit Salmonellen infiziert wurden, hatten eine höhere Kolonisierung und eine größere Ausscheidung der Infektionserreger. Da die beobachteten Veränderungen hauptsächlich mit lokalen angeborenen Immunantworten in Verbindung gebracht wurden, lag der Fokus dieser Doktorarbeit auf der Analyse direkter immunmodulatorischer Effekte von E. faecium SF68 auf intestinale Epithelzellen in einem in vitro Modell. NF-κB, einer der wichtigsten Regulatoren angeborener Immunantworten in Darmepithelzellen, die auf Mikrobiota und invasive enterische Pathogene treffen, wurde als Kriterium für die Bestimmung der Modulationen der Immunantwort in Darmepithelzellen ausgewählt. Das zellfreie Gesamtlysat von E. faecium SF68 hemmte die NF-κB-Aktivierung in intestinalen Epithelzellen, die von verschiedenen Wirtsspezies stammen. Dieser inhibitorische Effekt trat reversibel auf. Durch Entfernen des Lysats und Zugabe von frischem Kulturmedium erlangten die Zellen ihre NF-κB-Aktivität wieder. In Schweine-Darmepithelzellen, die mit dem probiotischen Lysat behandelt wurden, kam die phosphorylierte p65-Untereinheit von NF-κB an Serin 536 im Vergleich zu unbehandelten Zellen in geringerer Menge vor. Darüber hinaus deuteten fluoreszenzmikroskopische Aufnahmen von Schweine-Darmzellen auf eine Behinderung des Trafficking von NF-κB zwischen Zytoplasma und Zellkern hin. Zudem waren die mit dem bakteriellen Lysat behandelten Zellen nicht proliferativ, was mittels Zellproliferationsassay und Ki67-Färbung nachgewiesen wurde. Die Ammoniumsulfat-Fraktionierung von E. faecium SF68-Lysat und die Vergleichsanalyse der in den verschiedenen AS-Fraktionen vorhandenen Proteine ergab, dass mehrere am Arginin-Stoffwechsel beteiligte Enzyme in der aktiven AS-Fraktion vorhanden waren. Andere kommensale und klinische E. faecium-Isolate sowie Isolate anderer Enterokokken-Spezies, die Enzyme des Arginin-Deiminase-Pfads besitzen, einschließlich E. faecalis, E. hirae und E. durans, zeigten die gleiche inhibitorische Wirkung auf die NF-κB-Aktivität von IPEC-J2-Zellen. Die beiden Ausnahmen waren die Spezies E. gallinarum und E. casseliflavus. Diese beiden Spezies sind die einzigen motilen Enterokokken-Spezies, also Flagellin exprimieren. Flagellen sind ein starker Stimulator von Entzündungswegen, wie z.B. NF-κB, insbesondere in unserem In vitro Zellmodell. Darüber hinaus zeigten unsere Co-Inkubationsexperimente von Zellen mit E. faecium SF68-Lysat und verschiedenen TLR- und NLR-Liganden, dass die Verabreichung von Flagellin auf Zellen, die mit dem bakteriellen Lysat von E. faecium SF68 vorbehandelt wurden, die NF-κB-Aktivierung zusätzlich zu der inhibitorischen Wirkung des Lysats stimuliert. Lysat von Stämmen anderer Enterokokken-Spezies, die keine Enzyme des AD-Wegs enthalten, wie E. avium, E. cecorum und E. raffinosus, hingegen hemmten die NF-κB-Aktivität der behandelten IPEC-J2-Zellen nicht. Im nächsten Schritt wurde das arcA-Gen von E. faecium SF68, das Arginin-Deiminase exprimiert, in einen E. avium-Stamm kloniert, der keine hemmende Wirkung auf die NF-κB-Aktivität der Zellen besaß. E. avium, das die Arginin-Deiminase von E. faecium SF68 konstitutiv exprimiert, hatte die gleichen NF-κB-hemmenden Effekte auf porcine und humane intestinale Epithelzelllinien wie E. faecium SF68. Darüber hinaus zeigten Tests mit S. suis 10-Stamm aus anderen Bakteriengattungen, die AD-Weg-Enzyme besitzt, die gleichen Ergebnisse. Außerdem konnte die arcA-Knockout-Mutante von S. suis 10 die NF-κB-Aktivität der Zellen nicht beeinträchtigen. Schließlich wurde gezeigt, dass überexprimierte und gereinigte rekombinante Arginin-Deiminase von S. suis 10 die gleiche hemmende Wirkung hatte wie das zellfreie Gesamtlysat von S. suis 10 und E. faecium SF68. Die Arginin-Supplementierung des Kulturmediums wiederrum eliminierte die suppressiven Effekte auf die NF-κB-Aktivität, die durch Arginin-Deiminase oder bakterielle Lysate, die Arginin-Deiminase enthalten, vermittelt werden. Insgesamt postulieren wir, dass die entzündungshemmenden Effekte von E. faecium SF68 In vitro und möglicherweise auch In vivo mit dem Arginin-Deiminase-vermittelten Arginin-Abbau verbunden sind. Allerdings haben probiotische Bakterien im Allgemeinen eine große, komplizierte Anzahl von Molekülen und Metaboliten, wodurch sie mit intestinalen Epithel- und Immunzellen interagieren können. Daher können die immunmodulatorischen Effekte, die probiotische Mikroben im Darm hervorrufen, nicht auf einen einzigen Faktor zurückgeführt werden. Darüber hinaus können der Gesundheitszustand der Empfänger, ihr Alter und ihr Immunstatus für das Ergebnis der Verabreichung von nützlichen Mikroben ausschlaggebend sein. So sind zum Beispiel Neugeborene mit einem unterentwickelten Immunsystem, einer nicht vollständig ausgebeildeten Mikrobiota und einer höheren Stoffwechselaktivität empfindlicher gegenüber einer Arginin-Verarmung. Darüber hinaus kann das gleichzeitige Vorhandensein eines Darmpathogens, wie Salmonellen oder Giardien, die die Arginin-Depletion als Verteidigungsstrategie zur Behinderung der iNOS-Produktion und der Proliferation von Immunzellen im Wirt nutzen, den klinischen Zustand verschlimmern. Diese Erreger fördern den Arginin-Abbau durch ihre eigenen AD-Weg-Enzyme oder die Induktion von Arginin-verbrauchenden Enzymen in den Wirtszellen. Die Wirkungsmechanismen probiotischer Mikroben sollten gut und detailliert untersucht werden. Noch wichtiger ist, dass die Bewertungen und Entscheidungen über die Verabreichung einer probiotischen Mikrobe als therapeutische oder präventive Maßnahme in jedem Fall vorsichtig und individuell getroffen werden sollten, um den Einsatz dieser vorteilhaften biologischen Werkzeuge zu optimieren.

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum β-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.

Ascomycota account for about two-thirds of named fungal species.1 Over 98% of known Ascomycota belong to the Pezizomycotina, including many economically important species as well as diverse pathogens, decomposers, and mutualistic symbionts.2 Our understanding of Pezizomycotina evolution has until now been based on sampling traditionally well-defined taxonomic classes.3,4,5 However, considerable diversity exists in undersampled and uncultured, putatively early-diverging lineages, and the effect of these on evolutionary models has seldom been tested. We obtained genomes from 30 putative early-diverging lineages not included in recent phylogenomic analyses and analyzed these together with 451 genomes covering all available ascomycete genera. We show that 22 of these lineages, collectively representing over 600 species, trace back to a single origin that diverged from the common ancestor of Eurotiomycetes and Lecanoromycetes over 300 million years BP. The new clade, which we recognize as a more broadly defined Lichinomycetes, includes lichen and insect symbionts, endophytes, and putative mycorrhizae and encompasses a range of morphologies so disparate that they have recently been placed in six different taxonomic classes. To test for shared hidden features within this group, we analyzed genome content and compared gene repertoires to related groups in Ascomycota. Regardless of their lifestyle, Lichinomycetes have smaller genomes than most filamentous Ascomycota, with reduced arsenals of carbohydrate-degrading enzymes and secondary metabolite gene clusters. Our expanded genome sample resolves the relationships of numerous “orphan” ascomycetes and establishes the independent evolutionary origins of multiple mutualistic lifestyles within a single, morphologically hyperdiverse clade of fungi.

The class Dothideomycetes is the largest and most ecologically diverse class of fungi, comprising endophytes, epiphytes, saprobes, human and plant pathogens, lichens, and lichenicolous, nematode trapping and rock-inhabiting taxa. Members of this class are mainly characterized by bitunicate asci with fissitunicate dehiscence, and occur on broad range of hosts in aquatic and terrestrial habitats. Since the last monograph of families of Dothideomycetes in 2013, numerous novel species, genera, families and orders have been discovered. This has expanded information which has led to the modern classification in Dothideomycetes. In this paper, we provide a refined updated document on families of Dothideomycetes with emphasis on Dothideomycetidae and Pleosporomycetidae. We accept three orders with 25 families and four orders with 94 families in Dothideomycetidae and Pleosporomycetidae, respectively. The new family Paralophiostomataceae is introduced in Pleosporales. Each family is provided with an updated description, notes, including figures to represent the morphology, list of accepted genera, and economic and ecological significances. We also provide an overall phylogenetic tree of families in Dothideomycetes based on combined analysis of LSU, SSU, rpb-2 and tefl sequence data, and phylogenetic trees for each order in Dothideomycetidae and Pleosporomycetidae. Family-level trees are provided for the families which include several genera such as Mycosphaerellaceae and Teratosphaeriaceae. Two new genera (Ligninsphaeriopsis and Paralophiostoma) are introduced. Five new species (Biatrisopora borsei, Comoclathris galatellae, Ligninsphaeriopsis thailandica, Paralophiostoma hysterioides and Torula thailandica) are introduced based on morphology and phylogeny, together with nine new reports and seven new collections from different families.

Colonization of terrestrial environments by filamentous fungi relies on their ability to form networks that can forage for and connect resource patches. Despite the importance of these networks, ecologists rarely consider network features as functional traits because their measurement and interpretation are conceptually and methodologically difficult. To address these challenges, we have developed a pipeline to translate images of fungal mycelia, from both micro- and macro-scales, to weighted network graphs that capture ecologically relevant fungal behaviour. We focus on four properties that we hypothesize determine how fungi forage for resources, specifically: connectivity; relative construction cost; transport efficiency; and robustness against attack by fungivores. Constrained ordination and Pareto front analysis of these traits revealed that foraging strategies can be distinguished predominantly along a gradient of connectivity for micro- and macro-scale mycelial networks that is reminiscent of the qualitative ���phalanx��� and ���guerilla��� descriptors previously proposed in the literature. At one extreme are species with many inter-connections that increase the paths for multidirectional transport and robustness to damage, but with a high construction cost; at the other extreme are species with an opposite phenotype. Thus, we propose this approach represents a significant advance in quantifying ecological strategies for fungi using network information.

Tropical regions harbor a substantial diversity of lichenized fungi, but face numerous threats to their persistence, often even before previously unknown species have been described and their evolutionary relationships have been elucidated. Megalaria (Ramalinaceae) is a lichen-forming genus of fungi that produces crustose thalli, and includes a number of lineages occupying tropical rain forests; however, taxonomic and phylogenetic work on this clade is limited. Here we leverage both morphological and sequence data to describe a new species from the tropics, M. pachaylenophila. This taxon forms a crustose thallus, lacks secondary metabolites, and occurs in mangrove forests of Thailand. We supplemented molecular data from this species with data from other species, including two genera related to and occasionally included in Megalaria, namely Catillochroma and Lopezaria. Our analyses revealed Catillochroma species form a monophyletic group embedded within Megalaria, and we therefore recognize this clade at the subgeneric level. Since we only included the type species of Lopezaria in this study, we refrain from proposing a taxonomic conclusion for that clade at the moment. Several taxonomic combinations are made to reflect phylogenetic evidence supporting the inclusion of these species in Megalaria.

Six species of Sticta are described as new to science on the basis of material from Bolivia and supported by phylogenetic analysis of the fungal ITS barcoding marker. The species were resolved in all three of the clades (I, II, III) widespread and common in the Neotropics, as defined in an earlier study on the genus. Comparison with material from neighbouring countries (i.e. Colombia, Ecuador, Peru) suggests that these new species may be potentially endemic to the Bolivian Yungas ecoregion. For each species, a detailed morphological and anatomical description is given. Sticta amboroensis Ossowska, Kukwa, B. Moncada & Lücking is a medium-sized green-algal species with laminal to submarginal apothecia with hirsute margins and with light to dark brown lower tomentum. Sticta aymara Ossowska, Kukwa, B. Moncada, Flakus, Rodriguez-Flakus & Lücking is a comparatively small cyanobacterial taxon with Nostoc as photobiont, laminal, richly branched, aggregate isidia and a golden to chocolate-brown lower tomentum. The medium-sized, cyanobacterial S. bicellulata Ossowska, Kukwa, B. Moncada & Lücking has cyanobacterial photobiont, bicellular ascospores, apothecia with white to golden-brown hairs on the margins, K+ violet apothecial margin (ring around disc) and epihymenium and a white to dark brown lower tomentum. In contrast, the green-algal species, S. carrascoensis Ossowska, Kukwa, B. Moncada & Lücking is characterised by its large size, apothecia with dark brown hairs on the margins and a yellow medulla. The cyanobacterial S. catharinae Ossowska, B. Moncada, Kukwa, Flakus, Rodriguez-Flakus & Lücking forms stipitate thalli with Nostoc as photobiont, abundant, laminal to submarginal apothecia and a golden-brown lower tomentum. Finally, the cyanobacterial S. pseudoimpressula Ossowska, Kukwa, B. Moncada & Lücking produces laminal apothecia with an orange-yellow line of pruina along the margins which reacts K+ carmine-red. In addition to the six new Bolivian taxa, the cyanobacterial S. narinioana B. Moncada, Ossowska & Lücking is described as new from Colombia and it represents the closely-related sister species of the Bolivian S. aymara; it differs from the latter largely in the marginal instead of laminal isidia.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton–Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Human pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae, that cause life-threatening diseases and are resistant to many antibiotics, pose a serious health risk and, thus, a global social challenge. Therefore, further elucidation of bacterial resistance mechanisms to antimicrobial substances and the host immune defense is urgently required to improve existing treatment options and to develop new therapeutic approaches. The stringent response, which confers a non-specific stress resistance and facilitates the survival of bacteria, is induced in response to unfavorable environmental influences, such as nutrient starvation, by the accumulation of the alarmones guanosine tetra- or pentaphosphate ((p)ppGpp). While it was demonstrated that the stringent response is associated with increased antibiotic resistance, virulence and persistence, the underlying molecular mechanisms, particularly in Gram-positive bacteria, are still incompletely understood. Within this dissertation, it could be shown that (p)ppGpp is also essential for the oxidative stress resistance of S. aureus in the stationary phase (chapter 1). Quantitative analyses revealed higher respiratory chain activity and elevated total and free iron levels, causing increased intracellular levels of reactive oxygen species (ROS) in the (p)ppGpp0 mutant. Accordingly, the addition of iron chelators and antioxidants restored these physiological changes and mitigated the increased sensitivity of the (p)ppGpp0 mutant to oxidative stress and antibiotics. Thus, the maintenance of the intracellular iron and redox homeostasis was identified as a key mechanism of (p)ppGpp to promote increased stress resistance and antibiotic tolerance in S. aureus. The host immune system produces ROS and reactive chlorine species (RCS), such as H2O2 and hypochlorous acid (HOCl), as a central defense strategy. Consequently, the resistance of human pathogens to these reactive species is essential for their survival in the host. Low molecular weight thiols, such as glutathione (GSH) and bacillithiol (BSH), represent an important defense mechanism. Despite their function as antioxidants, they also protect thiol groups of proteins from irreversible over-oxidation through post-translational modification via S-thiolations. While the redox pathway for the regeneration of S-glutathionylated proteins has been fully elucidated, the equivalent process in Gram-positive Firmicutes, which use BSH instead of GSH, was incompletely understood. As presented in chapter 2, this work contributed to elucidate this further. It was demonstrated that the BSH redox pathway, consisting of BSH, bacilliredoxin A (BrxA) and the NADPH-dependent flavin oxidoreductase YpdA, is essential for the survival of S. aureus in the presence of H2O2 and HOCl. In addition, pathogens, such as S. aureus, encode for redox-sensing transcription factors, which utilize conserved cysteine residues to sense and respond to redox stress conditions via post-translational thiol modifications (chapter 3). Since these regulators control regulons that represent important enzymatic and non-enzymatic resistance mechanisms in bacteria, the work in hand focused on the identification and characterization of further transcription factors of the bacterial thiol-stress defense. For that purpose, RNA-seq analyses were used to investigate the bacterial stress response of S. pneumoniae to HOCl (chapter 4). The NmlR regulon was most strongly induced by HOCl stress and identified as an important resistance mechanism against oxidative stress and for the survival inside human macrophages. It consists of the nmlR and adhC genes that encode for the MerR-family transcriptional regulator NmlR and the Zn2+-dependent class III alcohol dehydrogenase AdhC. While NmlR was characterized previously as an aldehyde sensor, within this work, it was shown that NmlR also activates the transcription of the nmlR-adhC operon in response to oxidants. Molecular analyses revealed that the conserved cysteine (Cys52) is required for redox sensing by intermolecular disulfide formation and S-glutathionylation of NmlR. Although quinones are applied as potent antimicrobials since centuries, the bacterial resistance mechanisms are still incompletely resolved. Therefore, this work focussed on the characterization of the quinone stress response of S. aureus. The transcription profiles of the methylhydroquinone (MHQ) and lapachol stress responses revealed a high overlap of the provoked expression changes (chapters 5 and 6). For example, both quinones induced the oxidative stress response and the quinone-specific MhqR and QsrR regulons. Further analysis showed that, in contrast to MHQ, lapachol does not act as an electrophile but exerts its toxicity through the production of ROS (chapter 5). The antimicrobial effect of lapachol was oxygen dependent and could be reduced significantly by microaerophilic growth conditions. Phenotype analyses identified the H2O2 detoxifying catalase (KatA) and the BrxA/BSH/YpdA/NADPH redox pathway as important resistance mechanisms against lapachol stress in S. aureus. In contrast, the MhqR regulon conferred protection against MHQ but not lapachol. This finding suggests a substrate specificity of the enzymes MhqE and MhqD (chapter 6). Since the single cysteine residue (Cys95) of MhqR is not required for DNA binding and quinone sensing, it can be concluded that the MhqR repressor is not inactivated by a thiol-based mechanism but probably through ligand binding. In contrast, QsrR, another quinone-specific regulator of S. aureus, was shown to be regulated by different thiol switches (chapter 7). Unlike the MhqR regulon, the QsrR regulon was not only strongly induced by quinones but also by various oxidants. While QsrR was shown to sense oxidants by an intermolecular disulfide formation between the redox-active Cys4 and Cys29’, allicin caused the S-thioallylation of all three cysteine residues (Cys4, Cys29, Cys32) in vitro. Northern blot analyses indicated that the S-thioallylation of Cys4 is sufficient for the QsrR inactivation, while Cys4 and either Cys29’ or Cys32’ are required for the induction of the QsrR regulon by oxidants in vivo. Further transcriptional analysis and functional characterization revealed that the MhqR and QsrR regulons are also implicated in the resistance to various antibiotics, including ciprofloxacin and rifampicin (chapters 6 and 7). In addition, the ΔmhqR mutant showed an increased survival rate in long-term infection experiments with murine macrophages compared to the wild type, indicating that the MhqR regulon might function in persistence. Additionally, within the present work, the function of the GbaA regulon in the thiol stress response was examined (chapter 8). While the deletion of the SACOL2592-nmrA-2590 operon caused an enhanced susceptibility to diamide, allicin and aldehydes, the ΔgbaB mutant was impaired in its survival upon MHQ stress. It was further revealed that the conserved cysteines Cys55 and Cys104 of GbaA are required for the stress resistance against electrophiles. Substances that interfere with the bacterial redox homeostasis, either through the production of ROS, their electrophilic properties, or the inhibition of proteins, are currently developed and tested as alternatives to antibiotics (chapter 9). Especially ROS-generating substances represent promising treatment options since their non-specific mode of action minimizes the likelihood of resistance evolution. However, based on the usually lower cytotoxicity, specific inhibitors, e.g., of the low molecular weight thiol biosynthesis, might be more suitable for clinical application. Incited by the current global problem of the pandemic caused by the SARS-CoV-2 virus, the question of the antiviral activity of redox-based substances arose. Since the antimicrobial effect of allicin has been studied in detail, and previous studies suggested that allicin exerts an immunomodulatory effect, the antiviral activity of allicin on SARS-CoV-2 was investigated in a cell culture model within a collaboration project with the working group of Prof. Dr. Drosten (chapter 10). It was demonstrated that the administration of biocompatible allicin doses to SARS-CoV-2 infected cells reduced the viral RNA amount and the number of infectious viral particles by up to 70%. Using label-free quantitative proteomics, it was shown that SARS-CoV-2 causes the profound reprogramming of several host pathways, including gene expression and metabolism. In addition, a strong induction of the interferon signaling pathway and the interferon-stimulated gene signature was detected. Allicin treatment reverted several SARS-CoV-2 induced changes, including the expression of the interferon pathways, to levels of uninfected cells and reduced the expression of viral proteins significantly. The demonstrated antimicrobial activity of thiol-based substances suggests that they can be used in a modified form to combat infectious diseases. Lapachol, whose mode of action in S. aureus was elucidated in the present work, but also allicin, could serve as lead compounds to create more stable and less cytotoxic derivates. In addition, by combining global transcriptome analyses with molecular and microbiological assays, the present work contributed to the elucidation of bacterial adaptation strategies. In particular, the transcription factors NmlR, MhqR and QsrR were identified as important resistance mechanisms of the human pathogenic bacteria S. pneumoniae and S. aureus. Since the expression of these regulons is associated with increased bacterial resistance to the immune system and antibiotics, these proteins represent promising molecular targets for drug development.

Honey bees and wild bees are important pollinators and contribute to higher yields in agriculture and to the biodiversity of the natural fauna. However, globalization and industrialization of bee colonies lead to increasing infection pressure. Global trade and transport of colonies for maximizing the yield of a specific crop (e.g. almonds in the USA) leads to stress: Quick change of environments and environmental influences, no time to adapt, one-sided nutrition and high population densities of bee colonies from different parts of the world resulting in an interchange of diseases and parasites between the beehives, but also with the local pollinators. The most severe example is the mite Varroa destructor, which originates from Asia and is now found worldwide. Likewise, the pathogens that it vectors, e.g. deformed wing virus (DWV) affect the health of the bee colonies. Also from Asia is the originally Apis cerana parasitising honeybee pathogenic microsporidium N. ceranae, which parasitises Western honey bees Apis mellifera worldwide. Although it is one of the most common infections, publications on its epidemiology still seem to be contradictory. On the one hand N. ceranae is considered to be more virulent than N. apis and an emergent and deadly pathogen, on the other hand it has been described that there is no significant correlation between N. ceranae infection and colony losses. Within my dissertation I addressed several of these contradictions and tried to shed light on ambiguities. I investigated the prevalence of N. ceranae in Northeast Germany and was able to describe a seasonality, which has been ascribed only to N. apis before. Furthermore, I investigated the influence of N. ceranae infections occurring in autumn on the colony status of the spring of the following year and calculated its biological effect size. Finally, I wanted to investigate the so far unknown parameters of an N. ceranae infection in bumblebees (Bombus terrestris), which led to the conclusion that, contrary to numerous publications, bumblebees are not true hosts of N. ceranae.

Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi.

This habilitation thesis summarizes the results of 10 scientific publications (references: paper1-10) addressing the topic "Emergence of antimicrobial resistant bacteria in and beyond companion animal medicine”. The first chapter’s intention is to introduce my research area to the reader by providing basic information on the occurrence of antimicrobial resistance (AMR) in bacteria, including general resistance mechanisms, distribution and spread in different ecological environments and the commonly applied clinical classifications relevant for the veterinary medicine field. The following section characterizes the nature of health care associated infections (HAIs) in human and veterinary medicine, including baseline similarities and differences. Subsequently, bacterial pathogens exhibiting particular antibiotic resistances are introduced. Here, a strong focus is set on AMR bacteria frequently associated with HAIs in human and veterinary medicine, which are therefore also considered as “indicator pathogens”. Since β-lactams such as penicillins and cephalosporins are known for their excellent pharmacokinetic properties which is commonly accompanied by a low toxicity for mammals, this group of active substances is of particular importance for antimicrobial treatment of bacterial infectious diseases in both humans and animals. Infections with indicator pathogens harboring resistance towards β-lactams such as Staphylococcus aureus [methicillin resistant S. aureus, (MRSA)] and Escherichia coli [Extended-Spectrum β-Lactamase (ESBL)-producing E. coli (ESBL-E. coli)] are therefore often accompanied by treatment failure. Since antibiotics belonging to this group are among the “first choices” for empirical treatment of infectious diseases, including life-threatening scenarios such as septicemia, collaborative efforts to limit spread of β-lactam resistance are currently an important goal in human- and veterinary medicine. Additional resistances towards further classes of antibiotic substances are frequently associated with MRSA and ESBL-E. coli, leading to a further reduction of therapeutic possibilities in case of infection. In the second chapter, the publications’ results are summarized into four distinct sections. First, the importance of HAI in small animal and equine medicine is illustrated by a literature review considering outbreak events reported so far. A brief historical sketch reveals that problems due to HAI in animal clinics, including cases of zoonotic transmission, have been known since at least 1961. While there have been numerous reports in the past highlighting the technical and scientific developments during the last decades in companion-animal medicine, other important fields such as hygiene management, infection control and occupational safety of employees still lack comparable achievements. The current challenges faced by companion animal clinics are described by studies evaluating the introduction rates for multi drug resistant (MDR) indicator pathogens via colonized horses in a large German university clinic. Overall, n = 341 horses representing distinct medical indications (i.e. 233 horses with "colic symptoms" and 108 with "open wounds") were sampled immediately upon hospital admission. The overall screening results showed that MRSA was detected in 3.5% of the nasal swabs and ESBL-Enterobacteriaceae in 10.3% (mainly ESBL-E. coli) of the fecal samples. Subsequent molecular typing of the isolates showed that all MRSA belonged to sequence type (ST) 398, the currently predominating MRSA lineage identified in clinical samples of horse origin in Europe. In contrast, ESBL-E. coli showed a broad heterogeneity in genomic backgrounds associated with the β-lactam resistance. However, isolates belonging to ST complexes (STC) 1250 (31.7%) and STC10 (19.5%) clearly dominated the collection. Notably, all MRSA and ESBL- E. coli isolates isolated in these studies showed additional antimicrobial resistances to a minimum of three further classes of antibiotics. For assessment of a particular AMR situation in certain bacterial pathogens and/or environments, e.g. if surveillance of AMR in a pathogen causing HAI is needed, an unambiguous species identification is mandatory. Species identification is essential for sound interpretation of antimicrobial sensitivity testing (AST) results and therefore a prerequisite to classify the resistance phenotype of the pathogen correctly. Moreover, species identification is also needed for precise interpretation of additional results gained by other molecular biological methods. A combination of bacterial genome analysis with matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS) allowed us to differentiate between the species Staphylococcus intermedius, S. delphini and S. pseudintermedius, which was previously a time-consuming and uncertain approach. Since methicillin resistant S. pseudintermedius (MRSP) are often associated with resistances towards several other antimicrobials, a rapid and reliable species identification using MALDI-TOF MS promotes early and targeted antimicrobial therapy in cases of infection and, if necessary, the initiation of additional hygiene measures to prevent its spread. The following results demonstrate the overwhelming importance of constant adjustments of microbiological diagnostic screening methods for identification of MDR: In 2011, a novel variant of the methicillin resistance gene (later: mecC) was described for MRSA of cattle and human origin. A study we immediately initiated identified mecC-MRSA in clinical samples from horses, dogs, cats, wild animals and rodents. Here, the “weak” expression of the resistance phenotype, now known to be a common feature of mecC-MRSA, was a challenge to classify an isolate as MRS, which was achieved after diagnostic adjustments. As described above, methicillin resistance is often associated with other resistances, e.g. towards tetracyclines, zinc and arsenic compounds. Our detailed analysis of the integration site for the methicillin resistance-mediating mobile genetic element in staphylococci was identified as a "hot spot" for recombination events: We discovered a mosaic-like genomic structure which is prone to integration of mobile resistance-mediating elements of different types. The combination of up-to-date microbiological diagnostics with classical principles of infection epidemiology allowed us to answer important questions concerning possible transmission scenarios for MDR pathogens in companion animal clinics and identify risk factors for MRSA infections in these animals. In 2012, the ability of MRSA to cause HAI in companion animals and its importance as an indicator pathogen for veterinary medicine was already known. At that time however, similar information was not available for ESBL- E. coli. To address this deficit, we retrospectively investigated several cases of serious infections caused by ESBL-E. coli in hospitalized horses in a veterinary clinic. Comparative analysis of molecular typing results obtained for 13 ESBL-E. coli together with the horses’ clinical data revealed a putative spatio-temporal relationship of the isolates, which probably reflected several transmission events between these patients. In another case-control study, we have examined the possible risk factors for wound infections caused by MRSA in horses, dogs and cats. Multivariable logistic regression identified the following variables as risk factors for MRSA infection compared to methicillin susceptible infection: (i) the number of employees working at the veterinary setting (n>10; p, Diese Habilitationsschrift ist eine Zusammenfassung der Ergebnisse aus insgesamt 10 Publikationen mit dem Titel „Auftreten von Antibiotika-resistenten Bakterienspezies in der Kleintier- und Pferdemedizin und darüber hinaus“, welche aufgrund der jeweiligen inhaltlichen Schwerpunkte in vier Themenkomplexe untergliedert sind. Die den Ergebnissen vorangestellte Einleitung stellt das Forschungsgebiet insgesamt vor und bietet grundlegende Informationen zum Vorkommen von Antibiotikaresistenzen in Bakterien, deren Verbreitungswege sowie die für die Veterinärmedizin klinisch relevanten Klassifikationen. Es folgt eine kurze Einführung in das Thema „Health care“ assoziierte Infektionen (HAI) in der Human- und Veterinärmedizin, einschließlich einer Gegenüberstellung von grundsätzlichen Gemeinsamkeiten und Unterschieden. Im Anschluss werden die bakteriellen Pathogene mit Antibiotikaresistenzen vorgestellt, die von besonderer Bedeutung für HAIs in der Human- wie auch in der Veterinärmedizin sind und daher auch als „Indikatorpathogene“ gelten. Da β-Lactame wie Penicilline oder Cephalosporine im Allgemeinen hervorragende pharmakokinetische Eigenschaften besitzen bei gleichzeitig guter Verträglichkeit und geringer Toxizität, ist diese Wirkstoffgruppe besonders wichtig für die empirische Therapie von bakteriellen Infektionskrankheiten bei Menschen und Tieren. Resistenzen gegen β-Lactame in Indikatorpathogenen wie Staphylococcus aureus [Methicillin-resistente S. aureus, (kurz MRSA)] und Escherichia coli [(engl.) „Extended-Spectrum β-Lactamase“ (ESBL)- bildende E. coli (ESBL-E. coli)] wirken sich oftmals nachteilig auf den Behandlungserfolg aus, zumal Antibiotika dieser Gruppe für viele medizinische Indikationen Mittel der ersten Wahl darstellen. Zusätzliche Resistenzen gegenüber weiteren Wirkstoffklassen sind häufig mit diesen MRSA und ESBL-E. coli assoziiert, so dass sich die therapeutischen Möglichkeiten im Falle einer Erkrankung weiter verringern. Im 2. Kapitel werden die Ergebnisse der hier vorgestellten Publikationen in thematisch gegliederten Abschnitten zusammengefasst und eingeordnet. Zunächst wird die Bedeutung von HAI in der Kleintier- und Pferdemedizin durch Literatur zu Ausbruchsgeschehen mit den jeweils verantwortlichen Infektionserregern verdeutlicht. Diese Analyse zeigte, dass seit 1961 die Problematiken durch HAI in Tierkliniken sowie der Aspekt der möglichen wechselseitigen Übertragung von HAI-assoziierten Infektionserregern zwischen Mensch und Tier bekannt sind. Vielfach wurde in der Vergangenheit geschildert, dass im Hinblick auf Hygienemanagement, Infektionskontrolle sowie Arbeitsschutz der beschäftigten Mitarbeiter in der Veterinärmedizin ein vergleichsweise zu geringer Fortschritt zu verzeichnen ist. Mit welcher tatsächlichen Erregerlast Tierkliniken konfrontiert sein können, zeigen Ergebnisse zum Eintrag von multi-resistenten Infektionserregern (MRE) in eine große Deutsche Universitätspferdeklinik durch kolonisierte Pferde. Insgesamt wurden n= 341 Pferde, davon 233 mit dem Vorbericht „Kolik-Symptomatik“ und 108 mit dem Vorbericht „offene Wunde“ unmittelbar bei Klinikaufnahme beprobt. Die Screening-Ergebnisse insgesamt zeigten 3,5% MRSA-positive Nüsternabstriche sowie ESBL-Enterobacteriaceae in 10,3% (überwiegend ESBL-E. coli) der Kotproben. Die nachfolgende molekulare Typisierung der Isolate ergab, dass alle MRSA dem Sequenztyp (ST)398 zuzurechnen waren, der derzeit prädominanten Linie in Europa in Verbindung mit klinischen MRSA-Nachweisen beim Pferd. Bei den ESBL-E. coli zeigte die phylogenetische Analyse eine Vielzahl von unterschiedlichen Genotypen, es dominierten jedoch Isolate der ST Komplexe (STC)1250 (31,7%) und STC10 (19,5%). Alle MRSA und ESBL-E. coli Isolate zeigten zudem Resistenzen gegenüber mindestens drei weiteren Antibiotika-Wirkstoffklassen. Für die Bewertung der Resistenzsituation von Bakterien in beispielsweise einem bestimmten Umfeld, im Zusammenhang mit HAIs, oder für deren systematische Erfassung im Rahmen einer Surveillance, ist zunächst eine eindeutigen Speziesidentifizierung unerlässlich. Erst danach können die Ergebnisse der Empfindlichkeitstestung gegen Antibiotika und ggf. molekularbiologischen Ergebnisse sachgerecht für ein bestimmtes Isolat interpretiert werden. Durch Kombination von Genomanalyse mit (engl.) matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS) ist es gelungen, die für die veterinärmedizinische Diagnostik wichtige, eindeutige und schnelle Differenzierung zwischen den Spezies S. intermedius, S. delphini und S. pseudintermedius per MALDI-TOF MS vorzunehmen, die zuvor sehr zeitaufwendig und unsicher war. Da Methicillin-resistente S. pseudintermedius-Isolate (MRSP) häufig mit zahlreichen weiteren Resistenzen assoziiert sind und es Berichte über HAI durch MRSP in Kleintierkliniken gibt, ermöglicht nun die schnelle Diagnostik dieser Spezies per MALDI-TOF MS u.a. eine rasche und gezielte Therapie sowie ggf. die Einleitung von zusätzlichen Hygienemaßnahmen. Wie wichtig auch die ständige Anpassung der diagnostischen Screening-Methoden für MRE ist, zeigen die folgenden Ergebnisse: 2011 wurde erstmals eine neue Variante des Methicillin-Resistenzgens (später: mecC) bei MRSA von Rindern und Menschen beschrieben. Eine von uns umgehend eingeleitete Studie wies mecC-MRSA auch in klinischen Proben von Pferden, Hunden, Katzen, Wildtieren und Nagetieren nach, wobei die schwache Expression des Resistenzphänotyps bei mecC-MRSA eine Herausforderung für die Diagnostik insgesamt darstellt und Anpassungen erforderte. Methicilin-Resistenz ist häufig mit anderen Resistenzen, z.B. gegen Tetrazykline, Zink und Arsen, assoziiert. Unsere detaillierte Analyse der Integrationsstelle für das Methicillinresistenz-vermittelnde mobile genetische Element im Genom von Staphylokokken identifizierte einen „Hot-spot“ für Rekombinationsereignisse, wodurch diese Region häufig eine mosaikartige genomische Struktur aufweist, in die sich mobile, resistenzvermittelnde Elemente, insgesamt leicht integrieren können. Die Verbindung einer „up-to-date“ mikrobiologischen Diagnostik mit Methoden der klinischen Infektionsepidemiologie ermöglichte darüber hinaus die Beantwortung wichtiger Fragestellungen zu möglichen Transmissionsszenarien von MRE in Tierkliniken und die Identifikation von Risikofaktoren für MRE-Infektionen in der Kleintier- und Pferdemedizin – und- darüber hinaus. An den folgenden Beispielen soll dieser Zusammenhang beispielhaft erläutert werden. Während für MRSA 2012 bereits bekannt war, dass diese HAI Indikatorpathogene mit Ausbruchsgeschehen in Tierkliniken in Verbindung stehen können, war dies für ESBL-E. coli damals noch unklar. Daher haben wir mehrere Fälle von z.T. schweren Infektionen durch ESBL-E. coli bei hospitalisierten Pferden in einer Tierklinik retrospektiv untersucht. Durch die Analyse von molekularen Typisierungsergebnissen von 13 ESBL-E. coli mit den klinischen Daten der erkrankten Pferde konnte der wahrscheinliche spatio-temporale Zusammenhang der Isolate rekonstruiert werden, so dass es wahrscheinlich mehrere Transmissionsereignisse gab, die zu den Fällen führten. In einer weiteren Studie wurden die möglichen Risikofaktoren für eine Wundinfektion durch MRSA bei Pferden, Hunden und Katzen in einer Fall-Kontroll-Studie untersucht. Eine starke Korrelation für MRSA (n= 106) im Vergleich zu Methicillin-sensiblen S. aureus (n= 102) zeigte sich, wenn i) die Anzahl Beschäftigten in einer Praxis oder Klinik mindestens 10 war (p

In the recent past, the interest in microbiome research has increased because studies have suggested a strong link of microbial communities to their hosts. Along with a beneficial role of microbes, bacteria and viruses can have harmful effects and this pathogenic potential is of utmost interest. For public health, a robust and fast detection of pathogens is critical because it reduces the time for diagnosis and treatment and can lower the risk of transmission and mortality. This requires diagnostic assays to cover a broad spectrum for various potentially disease-causing agents. In the past, genome sequencing technologies have demonstrated both the immense variety of pathogens and the complexity of microbial communities. Reaching beyond the genetic potential that is addressed by metagenomics, metaproteomics enables investigating the metabolic and cellular pathways in which microbial enzymes are key players. Proteomics has reached a mature stage where it cannot only be used in a research context, but also for diagnostics as it had been demonstrated with newly emerging SARS-CoV-2 applications. This thesis aims at investigating the potential of using meta-omics technology and corresponding bioinformatics methods to process, analyze, and interpret data from experimental pathogen and microbiome samples. This work focuses on developing computational methods for detecting and characterizing bacterial and viral pathogens as well as for integrating host and microbiome data at different meta-omics levels. In addition, both experimental and bioinformatics benchmarking studies are performed. Finally, the aim is also to integrate methods and data for multiple omics levels, including metagenomics, metatranscriptomics, and metaproteomics.

The roles extracellular polymeric substances (EPS) play in mineral attachment and weathering were studied using genetically modified biofilms of the rock-inhabiting fungus Knufia petricola strain A95. Mutants deficient in melanin and/or carotenoid synthesis were grown as air-exposed biofilms. Extracted EPS were quantified and characterised using a combination of analytical techniques. The absence of melanin affected the quantity and composition of the produced EPS: mutants no longer able to form melanin synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, the melanin-producing strains attached more strongly to the mineral olivine and dissolved it at a higher rate. We hypothesise that the pullulan-related linkages, with their known adhesion functionality, enable fungal attachment and weathering. The released phenolic intermediates of melanin synthesis in the Δsdh1 mutant might play a role similar to Fe-chelating siderophores, driving olivine dissolution even further. These data demonstrate the need for careful compositional and quantitative analyses of biofilm-created microenvironments.

Seit den Untersuchungen von Robert Koch ist Bacillus anthracis als der Erreger von Milzbrand bekannt. Die verschiedenen St��mme sind genetisch sehr ��hnlich und besitzen spezifische chromosomale Marker sowie zwei Virulenzplasmide, pXO1 und pXO2, die die Synthese von Toxinen bzw. Kapsel kodieren. Es war darum eine ��berraschung, als ab 2001 der neuartige Erreger Bacillus cereus biovar anthracis (Bcbva) aus Menschenaffen und anderen S��ugetieren isoliert wurde, die in Regenwaldgebieten der Elfenbeink��ste (Stamm CI) und anderen afrikanischen L��ndern, u.a. in Kamerun (Stamm CA), an einer Milzbrand-��hnlichen Erkrankung verstorben waren. Bcbva kombiniert den chromosomalen Hintergrund von Bacillus cereus mit den beiden Virulenzplasmiden von B. anthracis und weist auf bakteriologischer Ebene Merkmale beider Spezies auf. Sowohl in B. anthracis als auch in Bcbva wird die Expression von Toxin- und Kapselgenen durch den pXO1-kodierten globalen Regulator AtxA kontrolliert und unter wirts��hnlichen Wachstumsbedingungen, die durch Bikarbonatzusatz im Medium und CO2-haltige Atmosph��re simuliert werden k��nnen, hochreguliert. In der vorliegenden Arbeit wurden die Prim��risolate Bcbva CI und CA mittels Genexpressionsanalysen und einer Sekretomanalyse n��her charakterisiert und mit zwei Vertretern von B. anthracis (Laborstamm B. anthracis Vollum und Wildisolat B. anthracis Dobichau) verglichen, wobei vor allem die Untersuchung der Virulenzmechanismen im Vordergrund stand. Es wurde eine Transkriptomanalyse (RNA-Sequenzierung) zum Vergleich der globalen Genexpression unter Umgebungsbedingungen und wirts��hnlichen Bedingungen mit jeweils drei biologischen Replikaten pro Stamm durchgef��hrt. Dabei wurden 24 Gene mit einem mindestens vierfachen Expressionsanstieg unter CO2-Bedingung bei allen vier St��mmen detektiert, darunter bekannte hochregulierte Virulenzgene wie die Kapselgene, wodurch die Validit��t der Ergebnisse best��tigt wurde. Plasmidgene wurden in CO2-Atmosph��re fast ausschlie��lich hoch- anstatt herunterreguliert, wobei die Anzahl pXO2-lokalisierter Gene die der pXO1-lokalisierten ��berstieg. Die Zahl der differentiell exprimierten Gene lag im Falle der hochregulierten Gene bei den Bcbva-St��mmen und bei B. anthracis Dobichau deutlich h��her als bei B. anthracis Vollum. F��r 41 Gene, darunter mehrere Gene f��r ABC-Transporter, wurde nur bei den beiden Bcbva-St��mmen, aber nicht bei B. anthracis, ein mindestens vierfacher Expressionsanstieg gezeigt. Hier gilt es zuk��nftig beispielsweise ��ber die Herstellung von Deletionsmutanten zu kl��ren, ob es sich bei den in Bcbva hochregulierten Genen um Virulenzfaktoren handelt. Die Analyse deckte weiterhin gravierende Unterschiede hinsichtlich der Anzahl an hoch- und herunterregulierten Genen zwischen den beiden Bcbva-Isolaten auf, die auf eine verschiedenartige Genregulation unter wirts��hnlichen Bedingungen hindeuten. Dies sollte in Untersuchungen mit weiteren Isolaten von Bcbva CI und CA ��berpr��ft werden, und mit Hilfe einer AtxA-Deletionsmutante k��nnte festgestellt werden, ob wirklich alle differentiell exprimierten Gene ��ber AtxA reguliert werden. Die massenspektrometrische Sekretomanalyse nach Anzucht unter wirts��hnlichen Bedingungen erm��glichte die Identifizierung von 33 potentiell diskriminierenden Proteinen, die spezifisch im ��berstand der untersuchten Bcbva-St��mme vorkamen. Hierzu z��hlte auch das pXO2-60 Protein, ein bekanntes Bcbva-spezifisches Antigen, dessen Nachweis die generierten Daten verifizierte. Es konnte allerdings kein weiteres Bcbva-spezifisches Antigen eindeutig detektiert werden. Obwohl teilweise geringe Sequenzidentit��ten zu B. anthracis vorliegen, besitzen alle Proteine eine fast identische Proteinsequenz zu Homologen aus anderen Spezies innerhalb der B. cereus-Gruppe und ihr Vorkommen ist weit verbreitet. Es bleibt offen, ob die betreffenden Proteine bei diesen Spezies ebenfalls sekretiert werden. Die Untersuchung der Genexpression w��hrend der Sporulation erfolgte mittels qPCR ausgew��hlter Sporulationsgene. Daf��r wurde RNA zu Beginn der station��ren Wachstumsphase sowie ein und zwei Stunden danach isoliert, und die Genexpression ��ber relative Quantifizierung gegen ein konstitutiv exprimiertes Haushaltsgen analysiert. Es konnte gezeigt werden, dass die hierarchisch ablaufende Genexpression w��hrend der Sporulation bei Bcbva und B. anthracis vergleichbar ist und dem allgemeinen Wissensstand ��ber die Versporung des Modellkeims B. subtilis entspricht. Die Initiation der Sporenbildung erfolgte in der vorliegenden Arbeit bei den untersuchten Bcbva-St��mmen eine Stunde fr��her, was auf eine tendenziell schnellere Reaktion auf n��hrstofflimitierende Umwelteinfl��sse bei Bcbva im Vergleich zu B. anthracis schlie��en l��sst. Bcbva besitzt im Gegensatz zu B. anthracis eine Insertion im Gen f��r den Sigmafaktor ��K. Das daf��r notwendige Ausschneiden der Insertion aus dem sigK-Gen scheint jedoch keinen limitierenden Einfluss auf die Kinetik der Versporung zu nehmen. Elektronenmikroskopisch wurde die schnellere Initiation der Sporenbildung bei Bcbva best��tigt, aber es konnten keine morphologischen Unterschiede der einzelnen Sporulationsstadien zwischen den beiden Spezies festgestellt werden. Die vorliegende Arbeit zeigt, dass die Genregulation bei den untersuchten St��mmen von Bcbva und B. anthracis weitgehend ��hnlich abl��uft, aber auch einige gravierende Unterschiede aufweist. Inwieweit das auf die verschiedenen ���Habitate��� der Erreger zur��ckzuf��hren ist, bedarf weiterer Kl��rung. Daf��r stellt diese Arbeit eine sehr gute Grundlage dar.

Staphylococcus (S.) aureus is a ubiquitous Gram-positive, spherical bacterium commonly isolated from humans, domestic-, livestock-, wildlife-, and even aquatic animals. The bacteria also survive and continue their growth on abiotic material in natural and artificial environments. Transmission of the immobile bacterium often occurs through direct contact with infected living beings or contaminated objects, whereby S. aureus is continuously forced to adapt to altered environmental conditions, including various host species. To overcome these challenges, members of the Genus Staphylococcus possess numerous strategies that include genomic alterations as well as the uptake or loss of mobile genetic elements (MGEs). The enormous adaptive capabilities and tendencies to accumulate genes encoding resistance traits define S. aureus as a pathogen of great clinical importance in human and veterinary medicine. Further insights into the adaptive strategies of this notorious pathogen are crucial, especially regarding the development of novel treatment options in case of infection. Against this background, changes in the essential regulatory quorum sensing (QS) system that promote bacterial niche adaptation were identified and characterised in this cumulative work. In addition, the genetic structure and putative role of the MGE mediating broad-spectrum β-lactam resistance in methicillin resistant S.aureus (MRSA) was investigated in detail using livestock-associated (LA)-MRSA as a prime example for adaptation to challenging environments. The data presented in Publication I revealed core genome alterations within the accessory gene regulator (agr) system, especially in the agrA and agrC genes, that led to variation or even silencing of the bacterial QS capabilities. Moreover, these changes occurred independently in strains with completely different genomic backgrounds of various animal origins across Europe as well as in closely related human isolates, clearly indicating the importance of QS regarding adaptive changes and not at least bacterial evolution. These adaptive modulatory changes enable the bacteria, beyond others, to survive among competing staphylococci belonging to the resident microbiota, simultaneously reducing their perceptibility by the host’s immune system while enhancing their biofilm formation capabilities. Consequently, S. aureus, lacking a functional agr system, saves the energy otherwise spend on QS and can conquer challenging environments since this deficit obviously promotes inter-species spread between different animal species, and, not at least, humans. The second part of this cumulative thesis focuses on a specific kind of MGEs, the Staphylococcal Cassette Chromosome (SCC) mec elements. A mechanism leading to the loss of a complete SCCmec element from its genomic integration site was reconstructed in detail for an MRSA sequence type (ST) 398 strain and described in Publication II. The investigated excision of the complete SCCmecVc element is suggested to be the result of homologous recombination within a repetitive sequence between the end of intergenic spacer region 1 and the first 48 bp of the large serine recombinases (ccr). Furthermore, the presence of an operon that encodes the machinery for autonomous replication of the deleted SCCmecVc element was revealed. Livestock-associated environmental conditions that are in particular associated with industrial pig farming were previously discussed as a potential trigger for the loss of SCCmec elements. To mimic conditions commonly associated with pig farming, LA-MRSA were exposed to porcine serum, an increased ammonia concentration and a combination of both for 10 min or 60 min. Since ccr genes are known for their capability to induce the elements’ uptake and loss, the growth conditions evaluated here did not significantly induce ccr transcription compared to the unchallenged controls. A detailed study of the deletion leading to the loss of a complete and functional SCCmec at defined genomic sites, on the other hand, strongly suggests a complex biological role of this particular event. While the induction of the ccr genes seems to depend on a massive cell- or DNA damage that leads to a strong activation of the SOS response, a far more efficient pathway for the horizontal spread of SCCmec is the occasional loss of complete elements at certain redundant “breaking points”, as described in Publication II. Since SCCmec elements possess the machinery for autonomous replication, this pathway might be of utmost epidemiological importance for the emergence, maintenance and spread of methicillin resistance. Both adaptation strategies investigated in this cumulative work highlight that the evolution of S. aureus will continue unabated. However, the unwearyingly evolution of these bacteria will lead to further problems in combating infections caused by S. aureus. The development of completely new treatment options, which consider the current knowledge on adaptation strategies of S. aureus, is therefore of clinical importance. Additionally, a holistic one-health approach that engages scientists from human and veterinary medicine as well as agricultural and environmental sciences is necessary to prevent further trans-sectoral transmission and spread by reducing the anthropogenic selective pressure that leads to resistance accumulation in bacteria., Staphylococcus (S.) aureus ist ein ubiquitär vorkommendes Gram-positives, kugelförmiges Bakterium, welches vielfach aus Proben von Menschen, Haus-, Nutz- und Wildtieren sowie von Meeressäugern isoliert wird. Das Bakterium kann auch auf abiotischem Material in natürlicher und künstlicher Umgebung überleben und dort sein Wachstum fortsetzen. Die Übertragung des unbeweglichen Bakteriums erfolgt häufig durch den direkten Kontakt mit infizierten Lebewesen oder kontaminierten Gegenständen, wodurch S.aureus ständig gezwungen ist, sich an veränderte Umweltbedingungen, einschließlich verschiedener Wirtsarten, anzupassen. Um diese Herausforderungen zu bewältigen, verfügen die Mitglieder des Genus Staphylococcus über zahlreiche Strategien, zu denen genomische Veränderungen sowie die Aufnahme oder der Verlust von mobilen genetischen Elementen (MGE) gehören. Die enormen Anpassungsfähigkeiten und die Tendenz zur Akkumulation von Genen, die für Resistenzeigenschaften kodieren, machen S. aureus zu einem Erreger von großer klinischer Bedeutung in der Human- und Veterinärmedizin. Weitere Erkenntnisse über die Anpassungsstrategien dieser Bakterien sind im Hinblick auf die Entwicklung neuer Behandlungsmöglichkeiten im Falle einer Infektion von entscheidender Bedeutung. Vor diesem Hintergrund wurden in dieser kumulativen Arbeit Veränderungen im regulatorisch wichtigen „Quorum-Sensing“ (QS)-System identifiziert und charakterisiert, welche die bakterielle Nischenanpassung unterstützen. Ergänzend dazu wurde die genetische Struktur und die mutmaßliche Rolle des MGE, welches die Breitspektrum-β-Lactam-Resistenz bei Methicillin-resistentem S. aureus (MRSA) vermittelt, eingehend untersucht, wobei hier der mit Nutztieren assoziierte (engl. livestock-associated (LA)) MRSA als Paradebeispiel für die Anpassung an schwierige Umgebungen dient. Die in der Publikation I vorgestellten Daten zeigen genomische Varianten innerhalb des akzessorischen Genregulatorsystems (agr), insbesondere in den agrA- und agrC-Genen, die zu einer Veränderung oder sogar Unterdrückung der bakteriellen QS-Fähigkeiten führten. Darüber hinaus traten diese Veränderungen unabhängig voneinander in Stämmen mit völlig unterschiedlichen phylogenetischem und geografischem Hintergrund, bei unterschiedlichen Tierarten sowie in eng verwandten menschlichen Isolaten auf, was eindeutig auf die Bedeutung von QS für adaptive Veränderungen und nicht zuletzt für die bakterielle Evolution hinweist. Diese adaptiven modulatorischen Veränderungen ermöglichen es den Bakterien, unter anderem unter konkurrierenden Staphylokokken der residenten Mikrobiota des Wirtes zu überleben und gleichzeitig ihre Perzeption durch das Immunsystem zu verringern, während die Fähigkeiten zur Biofilmbildung verstärkt werden. Infolgedessen verfügen S. aureus, denen ein funktionsfähiges agr-System fehlt, über einen energetischen Vorteil und können schwierige Umgebungen erobern, da dieses Defizit offensichtlich die Ausbreitung zwischen verschiedenen Tierarten und nicht zuletzt dem Menschen fördert. Der zweite Teil dieser kumulativen Arbeit konzentriert sich auf eine spezielle Art von MGEs, die „Staphylococcal Cassette Chromosome“ (SCC) mec-Elemente. Ein Mechanismus, der zum Verlust eines kompletten SCCmec-Elements von seiner genomischen Integrationsstelle führt, wurde für einen MRSA Sequenztyp (ST) 398-Stamm im Detail rekonstruiert und in Publikation II beschrieben. Die untersuchte Exzision des vollständigen SCCmecVc-Elements ist wahrscheinlich das Ergebnis homologer Rekombination innerhalb einer repetitiven Sequenz zwischen dem Ende der „intergenen Spacer“-Region 1 und den ersten 48 bp der großen Serin-Rekombinasen (ccr). Außerdem wurde das Vorhandensein eines Operons nachgewiesen, welches die Maschinerie für eine autonome Replikation des deletierten SCCmecVc-Elements kodiert. Mit der Tierhaltung einhergehende Umweltbedingungen, die insbesondere mit der industriellen Schweinehaltung assoziiert sind, wurden zuvor als möglicher Auslöser für den Verlust von SCCmec-Elementen diskutiert. Um Bedingungen nachzuahmen, die üblicherweise mit der Schweinehaltung in Verbindung gebracht werden, wurden LA-MRSA jeweils 10 Minuten oder 60 Minuten mit Schweineserum und einer erhöhten Ammoniakkonzentration sowie einer Kombination aus beidem behandelt. Die ccr-Gene sind dafür bekannt, dass sie die Aufnahme und den Verlust von Elementen induzieren können, jedoch führten die hier untersuchten Wachstumsbedingungen im Vergleich zu den unbelasteten Kontrollen zu keiner signifikanten Induktion der ccr-Transkription. Eine detaillierte Untersuchung des Vorgangs, welcher zum Verlust eines vollständigen und funktionsfähigen SCCmec an bestimmten Stellen im Genom führen kann, deutet dagegen auf eine komplexe biologische Rolle dieses Ereignisses hin. Während die Induktion der ccr-Gene von einer starken Aktivierung der SOS-Antwort in Folge einer massiven Zell- oder DNA-Schädigung abzuhängen scheint, ist ein weitaus effizienterer Weg für die horizontale Verbreitung von SCCmec der gelegentliche Verlust kompletter Elemente an bestimmten redundanten „Sollbruchstellen“, wie in Publikation II beschrieben. Da SCCmec-Elemente die Maschinerie zur autonomen Replikation besitzen, könnte dieser Weg von größter epidemiologischer Bedeutung für die Entstehung, Aufrechterhaltung und Verbreitung der Methicillin-Resistenz sein. Beide Anpassungsstrategien, die in dieser kumulativen Arbeit untersucht wurden, machen deutlich, dass die Evolution von S. aureus ungebremst weitergehen wird. Die unermüdliche Evolution dieser Bakterien wird jedoch zu weiteren Problemen bei der Bekämpfung von S. aureus verursachten Infektionen führen. Die Entwicklung völlig neuer Behandlungsmöglichkeiten, die das aktuelle Wissen über die Anpassungsstrategien von S. aureus berücksichtigen, ist daher von klinischer Bedeutung. Darüber hinaus sollte für die Zukunft ein ganzheitlicher „One-Health“-Ansatz in Betracht gezogen werden, der Wissenschaftlerinnen und Wissenschaftler aus der Human- und Veterinärmedizin sowie den Agrar- und Umweltwissenschaften einbezieht, um eine weitere sektorübergreifende Übertragung und Ausbreitung resistenter Bakterien zu vermindern, indem der anthropogene Selektionsdruck, welcher zu einer Resistenzakkumulation führt, verringert wird.

Every laboratory test needs validation by quality controls. For biocide susceptibility testing (BST), neither quality control (QC) strains nor QC ranges applicable to these strains are currently available. As QC strains, four well-defined laboratory reference strains (Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442), which have been used previously for biocide efficacy testing, were selected. In an interlaboratory trial with eleven participating laboratories, BST QC ranges should be developed for the aforementioned four strains and the four biocides benzalkonium chloride, chlorhexidine, octenidine and polyhexanide. The performance of three different lots of tryptic soy broth was explored using the broth microdilution method and the data were subsequently evaluated using the RangeFinder software. As a result, QC ranges were defined for all reference strain–biocide combinations, except for P. aeruginosa ATCC® 15442 with the two biocides chlorhexidine and polyhexanide. The development of the latter two QC ranges was not possible, due to the limited solubility of the biocides in the test range required for P. aeruginosa ATCC® 15442. The newly developed QC ranges comprise three to five dilution steps. The establishment of QC ranges will contribute to the validation of BST in the future.

Global ocean warming results in an increase of infectious diseases including an elevated emergence of Vibrio spp. in Northern Europe. The European Centre for Disease Prevention and Control reported annual periods of high to very high risks of infection with Vibrio spp. during summer months along the North Sea and Baltic Sea coasts. Based on those facts, the risk of Vibrio infections associated with recreational bathing in European coastal waters increases. To obtain an overview of the seasonal and spatial distribution of potentially human pathogenic Vibrio spp. at German coasts, this study monitored V. cholerae, V. parahaemolyticus, and V. vulnificus at seven recreational bathing areas from 2017 to 2018, including the heat wave event in summer 2018. The study shows that all three Vibrio species occurred in water and sediment samples at all sampling sites. Temperature was shown to be the main driving factor of Vibrio abundance, whereas Vibrio community composition was mainly modulated by salinity. A species-specific rapid increase was observed at water temperatures above 10°C, reaching the highest detection numbers during the heat wave event with abundances of 4.5 log10 CFU+1/100 ml of seawater and 6.5 log10 CFU+1/100 g of sediment. Due to salinity, the dominant Vibrio species found in North Sea samples was V. parahaemolyticus, whereas V. vulnificus was predominantly detected in Baltic Sea samples. Most detections of V. cholerae were associated with estuarine samples from both seas. Vibrio spp. concentrations in sediments were up to three log higher compared to water samples, indicating that sediments are an important habitat for Vibrio spp. to persist in the environment. Antibiotic resistances were found against beta-lactam antibiotics (ampicillin 31%, cefazolin 36%, and oxacillin and penicillin 100%) and trimethoprim-sulfamethoxazole (45%). Moreover, isolates harboring pathogenicity-associated genes such as trh for V. parahaemolyticus as well as vcg, cap/wcv, and the 16S rRNA-type B variant for V. vulnificus were detected. All sampled V. cholerae isolates were identified as non-toxigenic non-O1/non-O139 serotypes. To sum up, increasing water temperatures at German North Sea and Baltic Sea coasts provoke elevated Vibrio numbers and encourage human recreational water activities, resulting in increased exposure rates. Owing to a moderate Baltic Sea salinity, the risk of V. vulnificus infections is of particular concern.

Pathogens 11(12), 1455 (2022). doi:10.3390/pathogens11121455 special issue: "Special Issue "What Does Not Kill Them, Makes Them Stronger: Is the Inappropriate Use of Biocides a Driver of Antimicrobial Resistance?" / Special Issue Editors: Prof. Dr. Sascha Al Dahouk, Dr. Ralf Dieckmann", Published by MDPI, Basel

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125–241 kbp contain 70–170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Plasmids are mobile genetic elements, contributing to the spread of resistance determinants by horizontal gene transfer. Plasmid-mediated quinolone resistances (PMQRs) are important determinants able to decrease the antimicrobial susceptibility of bacteria against fluoroquinolones and quinolones. The PMQR gene qnrS1, especially, is broadly present in the livestock and food sector. Thus, it is of interest to understand the characteristics of plasmids able to carry and disseminate this determinant and therewith contribute to the resistance development against this class of high-priority, critically important antimicrobials. Therefore, we investigated all commensal Escherichia (E.) coli isolates, with reduced susceptibility to quinolones, recovered during the annual zoonosis monitoring 2017 in the pork and beef production chain in Germany (n = 2799). Through short-read whole-genome sequencing and bioinformatics analysis, the composition of the plasmids and factors involved in their occurrence were determined. We analysed the presence and structures of predominant plasmids carrying the PMQR qnrS1. This gene was most frequently located on IncX plasmids. Although the E. coli harbouring these IncX plasmids were highly diverse in their sequence types as well as their phenotypic resistance profiles, the IncX plasmids-carrying the qnrS1 gene were rather conserved. Thus, we only detected three distinct IncX plasmids carrying qnrS1 in the investigated isolates. The IncX plasmids were assigned either to IncX1 or to IncX3. All qnrS1-carrying IncX plasmids further harboured a β-lactamase gene (bla). In addition, all investigated IncX plasmids were transmissible. Overall, we found highly heterogenic E. coli harbouring conserved IncX plasmids as vehicles for the most prevalent qnr gene qnrS1. These IncX plasmids may play an important role in the dissemination of those two resistance determinants and their presence, transfer and co-selection properties require a deeper understanding for a thorough risk assessment.

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.

Many antibiotic resistance genes present in human pathogenic bacteria are believed to originate from environmental bacteria. Conjugation of antibiotic resistance conferring plasmids is considered to be one of the major reasons for the increasing prevalence of antibiotic resistances. A hotspot for plasmid-based horizontal gene transfer is the phyllosphere, i.e., the surfaces of aboveground plant parts. Bacteria in the phyllosphere might serve as intermediate hosts with transfer capability to human pathogenic bacteria. In this study, the exchange of mobilisable and self-transmissible plasmids via conjugation was evaluated. The conjugation from the laboratory strain Escherichia coli S17-1, the model phyllosphere coloniser Pantoea eucalypti 299R, and the model pathogen E. coli O157:H7 to the recipient strain E. coli O157:H7::MRE103 (EcO157:H7red) in the phyllosphere of Arabidopsis thaliana was determined. The results suggest that short-term occurrence of a competent donor is sufficient to fix plasmids in a recipient population of E. coli O157:H7red. The spread of self-transmissible plasmids was limited after initial steep increases of transconjugants that contributed up to 10% of the total recipient population. The here-presented data of plasmid transfer will be important for future modelling approaches to estimate environmental spread of antibiotic resistance in agricultural production environments.

Brucellosis is a bacterial zoonotic disease that affects different animal species and can be spread to humans via direct contact as well as via the food chain. Although brucellosis is a serious health hazard, its significance has largely been neglected in many countries. However, brucellosis has been the subject of interest in developing countries, especially in Pakistan where this disease is endemic. Brucellosis is a puzzling disease in terms of taxonomy and typing, clinical signs, pathobiology, diagnosis, vaccination and treatment. Practical eradication programs include test and slaughter policies and vaccination as preventive measures in animals. The purpose of this review was to provide an updated and comprehensive insight into brucellosis in Pakistan with particular emphasis on its occurrence in humans and various animal species, the diagnostic tests applied as well as prevention, treatment and eradication aspects.

Fungus-bacterium interactions are widespread, encompass multiple interaction types from mutualism to parasitism, and have been frequent targets for microbial inoculant development. In this study, using in vitro systems combined with confocal laser scanning microscopy and real-time quantitative PCR, we test whether the nitrogen-fixing bacterium Kosakonia radicincitans can provide protection to the plant-beneficial fungus Serendipita indica, which inhabits the rhizosphere and colonizes plants as an endophyte, from the fungus-feeding bacterium Collimonas fungivorans. We show that K. radicincitans can protect fungal hyphae from bacterial feeding on solid agar medium, with probable mechanisms being quick hyphal colonization and biofilm formation. We furthermore find evidence for different feeding modes of K. radicincitans and C. fungivorans, namely “metabolite” and “hyphal feeding”, respectively. Overall, we demonstrate, to our knowledge, the first evidence for a bacterial, biofilm-based protection of fungal hyphae against attack by a fungus-feeding, bacterial predator on solid agar medium. Besides highlighting the importance of tripartite microbial interactions, we discuss implications of our results for the development and application of microbial consortium-based bioprotectants and biostimulants.

Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant β-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-β-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.

The increasing number of new fungal species described from all over the world along with the use of genetics to define taxa, has dramatically changed the classification system of early-diverging fungi over the past several decades. The number of phyla established for non-Dikarya fungi has increased from 2 to 17. However, to date, both the classification and phylogeny of the basal fungi are still unresolved. In this article, we review the recent taxonomy of the basal fungi and re-evaluate the relationships among early-diverging lineages of fungal phyla. We also provide information on the ecology and distribution in Mucoromycota and highlight the impact of chytrids on amphibian populations. Species concepts in Chytridiomycota, Aphelidiomycota, Rozellomycota, Neocallimastigomycota are discussed in this paper. To preserve the current application of the genus Nephridiophaga (Chytridiomycota: Nephridiophagales), a new type species, Nephridiophaga blattellae, is proposed.

Bartonellae are facultative intracellular alpha-proteobacteria often transmitted by arthropods. Ixodes ricinus is the most important vector for arthropod-borne pathogens in Europe. However, its vector competence for Bartonella spp. is still unclear. This study aimed to experimentally compare its vector competence for three Bartonella species: B. henselae, B. grahamii, and B. schoenbuchensis. A total of 1333 ticks (1021 nymphs and 312 adults) were separated into four groups, one for each pathogen and a negative control group. Ticks were fed artificially with bovine blood spiked with the respective Bartonella species. DNA was extracted from selected ticks to verify Bartonella-infection by PCR. DNA of Bartonella spp. was detected in 34% of nymphs and females after feeding. The best engorgement results were obtained by ticks fed with B. henselae-spiked blood (65.3%) and B. schoenbuchensis (61.6%). Significantly more nymphs fed on infected blood (37.3%) molted into adults compared to the control group (11.4%). Bartonella DNA was found in 22% of eggs laid by previously infected females and in 8.6% of adults molted from infected nymphs. The transovarial and transstadial transmission of bartonellae suggest that I. ricinus could be a potential vector for three bacteria.

We describe the new lichenized fungus Lasioloma antillarum Lucking, Hognabba & Sipman from the Netherlands Antilles. The new species is characterized by a corticolous growth habit, apothecia with shortly tomentose margins, and rather small (35–50 × 12–16 µm), muriform ascospores in numbers of 2(–4) per ascus. The material had originally been identified as Calopadia phyllogena (Mull. Arg.) Vězda, with associated sequence data, but in phylogenetic analyses consistently fell outside the latter genus. Its revised identification as a species of Lasioloma is consistent with its phylogenetic position and underlines the necessity of posterior annotations in public sequence repositories, in order to correct previous identifications. Citation: Lucking R., Hognabba F. & Sipman H. J. M. 2021: Lasioloma antillarum (Ascomycota: Pilocarpaceae), a new lichenized fungus from the Antilles, and the importance of posterior annotations of sequence data in public repositories. – Willdenowia 51: 83–89. Version of record first published online on 23 March 2021 ahead of inclusion in April 2021 issue.

Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Skłodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals. ispartof: VIRUSES-BASEL vol:13 issue:5 ispartof: location:Switzerland status: published

Antimicrobial susceptibility testing (AST) should be conducted in a standardized manner prior to the start of an antimicrobial treatment. For fastidious bacteria, such as porcine Mycoplasma (‘Mesomycoplasma’) spp., specifically M. hyorhinis, neither guidelines or standards for the performance of AST, nor quality control strains for the validation of AST results are approved by organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CLSI- and EUCAST-approved quality control strains Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were chosen to validate AST by broth microdilution using modified Friis broth, developed as growth medium for porcine Mycoplasma (‘Mesomycoplasma’) spp. The antimicrobial agents doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin were examined using customized SensititreTM microtiter plates. Minimal inhibitory concentrations, determined after 24, 48, and 72 h, were mostly within the CLSI-approved quality control ranges for defined antimicrobial agents. We propose the use of the combination of E. faecalis ATCC 29212 and S. aureus ATCC 29213 as surrogate quality control strains for the validation of future AST results obtained for M. hyorhinis by broth microdilution using modified Friis broth.

V. cholerae bacteria are distributed in aquatic environments with low to moderate salinity throughout the world. Toxigenic serogroup O1/O139 V. cholerae strains can cause the epidemic disease cholera. In northern European waters, non-O1, non-O139 V. cholerae strains occur, some of which have been associated with gastrointestinal or extraintestinal infections. So far, infections caused by these bacteria are rare in Germany. However, due to climate change, an increase in non-O1, non-O139 V. cholerae prevalence is expected. Given the popularity of German coastal regions as a travel destination, an increased exposure of the population to strains of these serogroups must be assumed for future years. Therefore, by comparing MLST sequence types (ST) and virulence factor profiles of environmental isolates and clinical isolates, a first risk assessment for non-O1, non-O139 V. cholerae strains from German coastal waters was carried out in this work. The similarity of the virulence gene profiles of environmental strains and clinical strains - irrespective of the ST - as well as the hemolytic activity against human erythrocytes could indicate a human pathogenic potential of many environmental strains in the North Sea and Baltic Sea. Data comparison to a first follow-up study conducted in the German Bight suggests that environmental changes resulting from climate change possibly foster V. cholerae strains with a higher infective risk. The whole genome sequencing (WGS)-based typing scheme that was developed and established at the Vibrio consiliary laboratory in the context of this dissertation can serve as a tool for monitoring and deeper investigation of virulence gene profiles of future non-O1, non-O139 V. cholerae isolates obtained from public health-relevant sources. While Vibrio species such as V. cholerae have been intensively studied for decades as a result of the global disease burden, other Vibrio species such as the presumed environmental species V. navarrensis are hardly explored. Recently, V. navarrensis strains were detected in human clinical specimens as well as veterinary abortion specimens from livestock in Germany. For identification of new potential environmental reservoirs in Germany and against the background of a possible zoonotic potential, comparative genotypic analyses of V. navarrensis isolates from environmental, veterinary, and human clinical specimens were performed in this work. The results of SNP and MLSA analyses suggest the occurrence of V. navarrensis in German freshwater environments, which would extend the range of this species from coastal waters of the North Sea and Baltic Sea to inland waters in Germany. Ingestion of V. navarrensis by farm animals through uptake of surface water seems possible. The presence of an MLSA subcluster encompassing all veterinary and human clinical strains could indicate a human pathogenic potential of the former. The design of a WGS-based analytical procedure for typing of virulence-associated genes in V. navarrensis isolates provided deeper insights into the genetic makeup of all strains. Phenotypic traits such as the hemolytic activity of the investigated environmental and veterinary strains against sheep erythrocytes and human red blood cells underline the pathogenic character of this Vibrio species. A new case of a human infection in summer 2020 supports this assessment. The developed V. navarrensis-specific vvhA and tlh singleplex PCRs represent promising tools for rapid species diagnostics and may thereby facilitate future monitoring and research studies on Vibrio bacteria in the context of the “One Health” approach., V. cholerae-Bakterien sind in aquatischen Umwelten mit geringer bis moderater Salinität weltweit verbreitet. Toxigene V. cholerae-Stämme der Serogruppe O1/O139 können die epidemische Erkrankung Cholera verursachen. In nordeuropäischen Gewässern kommen non-O1, non-O139 V. cholerae-Stämme vor, von denen einige mit gastrointestinalen oder extraintestinalen Infektionen in Verbindung gebracht wurden. Bislang sind durch diese Bakterien verursachte Infektionen in Deutschland selten. Aufgrund des Klimawandels wird jedoch ein Anstieg in der non-O1, non-O139 V. cholerae-Prävalenz erwartet. Angesichts der Beliebtheit deutscher Küstenregionen als Reiseziel muss in den kommenden Jahren von einer erhöhten Exposition der Bevölkerung gegenüber Stämmen dieser Serogruppen ausgegangen werden. Daher wurde in dieser Arbeit über den Vergleich von MLST-Sequenztypen (ST) und Virulenzfaktorprofilen von Umweltisolaten und klinischen Isolaten eine erste Risikobewertung für non-O1, non-O139 V. cholerae-Stämme aus deutschen Küstengewässern durchgeführt. Die Ähnlichkeit der Virulenzgenprofile von Umweltstämmen und klinischen Stämmen - unabhängig vom ST - sowie die hämolytische Aktivität gegenüber humanen Erythrozyten könnten auf ein humanpathogenes Potential vieler Umweltstämme in der Nordsee und Ostsee hindeuten. Ein Vergleich der Daten mit denen einer ersten Folgestudie, die in der Deutschen Bucht durchgeführt wurde, legt nahe, dass sich aus dem Klimawandel ergebende Umweltveränderungen möglicherweise V. cholerae-Stämme mit einem höheren Infektionsrisiko fördern. Das whole genome sequencing (WGS)-basierte Typisierungsschema, das im Rahmen dieser Dissertation entwickelt und am Vibrio-Konsiliarlabor etabliert wurde, kann als Werkzeug zur Überwachung und tieferen Untersuchung von Virulenzgenprofilen künftiger non-O1, non-O139 V. cholerae-Isolate aus für die öffentliche Gesundheit relevanten Quellen dienen. Während Vibrio-Arten wie V. cholerae infolge der globalen Krankheitslast seit Jahrzehnten intensiv untersucht werden, sind andere Vibrio-Arten wie die mutmaßliche Umweltspezies V. navarrensis kaum erforscht. Kürzlich wurden V. navarrensis-Stämme in humanen klinischen Proben sowie veterinären Abortproben aus Nutztieren in Deutschland nachgewiesen. Zur Identifizierung neuer potentieller Umweltreservoire in Deutschland und vor dem Hintergrund eines möglichen zoonotischen Potentials wurden in dieser Arbeit vergleichende genotypische Analysen von V. navarrensis-Isolaten aus Umweltproben, Veterinärproben und humanen klinischen Proben durchgeführt. Die Ergebnisse der SNP- und MLSA-Analysen deuten auf das Vorkommen von V. navarrensis in deutschen Süßwasserumwelten hin, was das Verbreitungsgebiet dieser Spezies von Küstengewässern der Nord- und Ostsee auf Binnengewässer in Deutschland erweitern würde. Eine Aufnahme von V. navarrensis über Oberflächenwasser-Aufnahme durch Nutztiere scheint möglich. Das Vorliegen eines MLSA-Subclusters, welches alle veterinären und humanen klinischen Stämme umfasst, könnte auf ein humanpathogenes Potential der Ersteren hinweisen. Das Design eines WGS-basierten analytischen Verfahrens zur Typisierung Virulenz-assoziierter Gene in V. navarrensis-Isolaten bot tiefere Einblicke in die genetische Ausstattung aller Stämme. Phänotypische Merkmale wie die hämolytische Aktivität der untersuchten Umwelt- und Veterinärstämme gegenüber Schaferythrozyten und humanen Erythrozyten betonen den pathogenen Charakter dieser Vibrio-Art. Ein neuer Fall einer Humaninfektion im Sommer 2020 stützt diese Bewertung. Die entwickelten V. navarrensis-spezifischen vvhA- und tlh-Singleplex-PCRs stellen vielversprechende Werkzeuge für eine schnelle Speziesdiagnostik dar und dürften dadurch künftige Überwachungs- und Forschungsstudien zu Vibrio-Bakterien im Rahmen des “One Health”-Ansatzes erleichtern.

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals.

The present work describes a new computer-assisted image analysis method for the rapid, simple, objective and reproducible quantification of actively discharged fungal spores which can serve as a manual for laboratories working in this context. The method can be used with conventional laboratory equipment by using bright field microscopes, standard scanners and the open-source software ImageJ. Compared to other conidia quantification methods by computer-assisted image analysis, the presented method bears a higher potential to be applied for large-scale sample quantities. The key to make quantification faster is the calculation of the linear relationship between the gray value and the automatically counted number of conidia that has only to be performed once in the beginning of analysis. Afterwards, the gray value is used as single parameter for quantification. The fast, easy and objective determination of sporulation capacity enables facilitated quality control of fungal formulations designed for biological pest control.•Rapid, simple, objective and reproducible quantification of fungal sporulation suitable for large-scale sample quantities.•Requires conventional laboratory equipment and open-source software without technical or computational expertise.•The number of automatically counted conidia can be correlated with the gray value and after initial calculation of a linear fit, the gray value can be applied as single quantification parameter., Graphical abstract Image, graphical abstract

Sticta is a subcosmopolitan genus most diverse in the tropics. Traditionally, many taxa were considered to be widespread and morphologically variable, following broadly circumscribed morphodemes. Among these is the S. weigelii morphodeme, characterized by a cyanobacterial photobiont and rather narrow, flabellate to truncate or tapering lobes producing predominantly marginal isidia. Molecular phylogenetic analyses focusing on the ITS fungal barcoding marker revealed that this morphodeme represents several species, some of which are only distantly related to each other. Here we describe two species and one subspecies of this morphodeme as new to science, based on analysis of 400 specimens, for 344 of which we generated ITS barcoding data. The two new species, S. andina and S. scabrosa, are broadly distributed in the Neotropics and also found in Hawaii, where the latter is represented by the new subspecies, S. scabrosa subsp. hawaiiensis; in the case of S. andina, the species is also found in the Azores. Sticta andina exhibits high phenotypic variation and reticulate genetic diversification, whereas the phenotypically rather uniform S. scabrosa contains two main haplotypes, one restricted to Hawaii. Sticta andina occurs in well-preserved montane to andine forests and paramos, whereas the two subspecies of S. scabrosa are found in tropical lowland to lower montane forests, tolerating disturbance and extending into anthropogenic habitats.Citation: Moncada B., Mercado-Diaz J. A., Smith C. W., Bungartz F., Serusiaux E., Lumbsch H. T. & Lucking R. 2021: Two new common, previously unrecognized species in the Sticta weigelii morphodeme (Ascomycota: Peltigeraceae). – Willdenowia 51: 35–45.Version of record first published online on 24 February 2021 ahead of inclusion in April 2021 issue.

Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.

Marine mammals are sentinels for the marine ecosystem and threatened by numerous factors including infectious diseases. One of the most frequently isolated bacteria are beta-hemolytic streptococci. However, knowledge on ecology and epidemiology of streptococcal species in marine mammals is very limited. This review summarizes published reports on streptococcal species, which have been detected in marine mammals. Furthermore, we discuss streptococcal transmission between and adaptation to their marine mammalian hosts. We conclude that streptococci colonize and/or infect marine mammals very frequently, but in many cases, streptococci isolated from marine mammals have not been further identified. How these bacteria disseminate and adapt to their specific niches can only be speculated due to the lack of respective research. Considering the relevance of pathogenic streptococci for marine mammals as part of the marine ecosystem, it seems that they have been neglected and should receive scientific interest in the future.

It is now a decade since The International Commission on the Taxonomy of Fungi (ICTF) produced an overview of requirements and best practices for describing a new fungal species. In the meantime the International Code of Nomenclature for algae, fungi, and plants (ICNafp) has changed from its former name (the International Code of Botanical Nomenclature) and introduced new formal requirements for valid publication of species scientific names, including the separation of provisions specific to Fungi and organisms treated as fungi in a new Chapter F. Equally transformative have been changes in the data collection, data dissemination, and analytical tools available to mycologists. This paper provides an updated and expanded discussion of current publication requirements along with best practices for the description of new fungal species and publication of new names and for improving accessibility of their associated metadata that have developed over the last 10 years. Additionally, we provide: (1) model papers for different fungal groups and circumstances; (2) a checklist to simplify meeting (i) the requirements of the ICNafp to ensure the effective, valid and legitimate publication of names of new taxa, and (ii) minimally accepted standards for description; and, (3) templates for preparing standardized species descriptions.

Human listeriosis is a comparatively rare but serious infectious disease. The high hospitalisation and mortality rate make it a major public health concern worldwide. Elderly and immunocompromised people as well as pregnant women are at increased risk of infection. Listeriosis is caused by consumption of food contaminated with the bacterium Listeria (L.) monocytogenes. Listeria monocytogenes is widespread in the environment and in animals. It enters the food chain either via contaminated raw animal or plant products or via cross-contamination during food processing. The overall aim of the present dissertation was to prevent cases of listeriosis in humans using state-of-the-art molecular epidemiological methods and genetic profiling of bacterial isolates. As a support to classical epidemiology, molecular typing methods are used to monitor the entry and spread of L. monocytogenes along the food chain up to the consumer. In recent years, the possibility of whole genome sequencing (WGS) has revolutionised the molecular typing of bacterial isolates. Based on the knowledge gained in the present doctoral project, the previous gold standard for fine typing, pulsed-field electrophoresis, was completely replaced by WGS in the National Reference Laboratory (NRL) for L. monocytogenes in 2018. As part of the present project, the value of WGS as a high-resolution molecular surveillance tool for L. monocytogenes has been widely communicated to stakeholders along the food chain. Based on the protocols established in the NRL during the doctoral project, the tool is now available to the national monitoring authorities. Within the dissertation project, the bioinformatic analysis of WGS data of L. monocytogenes was optimised for data exchange, a listeriosis outbreak and the spread of Listeria spp. in a food processing plant were investigated, and the significance of the European Rapid Alert System for Food and Feed (RASFF) for foodborne outbreaks was evaluated. Using WGS, contamination and infection chains can be traced with unprecedented precision and thus be stopped. However, the methods for analysing WGS data are diverse and not yet fully harmonised, which hinders data sharing. In the present work, it was shown that different WGS analysis methods for L. monocytogenes generate largely comparable results. A translation code developed within this doctoral project allows information on clusters to be exchanged between sectors (e.g. food safety, public health) and countries even without harmonised methods. This approach offers a major advantage as it allows rapid communication between stakeholders, especially in time-critical situations such as listeriosis outbreaks. An important prerequisite for the prevention of listeriosis infections is a stronger focus on industrial hygiene in food processing. This became clear in the example of a study of a poultry processing chain carried out as part of this doctoral project. By means of WGS typing, it could be shown that Listeria spp. are transferred from the animal and from the environment to the finished food product. In addition, bacteria were shown to spread to surfaces in the production environment, favouring dangerous cross-contamination. Since the introduction of WGS for typing L. monocytogenes in Germany, in cross-sectoral collaboration, a large number of listeriosis outbreaks could be clarified and stopped. This work provides insight into a large national listeriosis outbreak lasting several years, which was lead investigated in the present doctoral project. Using WGS typing, the outbreak was traced back to ready-to-eat meat products and their producer. By using forward checking as a supplement to the usual backtracking in outbreak investigations, two different clusters of listeriosis cases could be assigned to the same producer. Only through such a two-sided control strategy, combining backtracking and forward checking, can listeriosis outbreaks be significantly minimised in the future. As a result of the intensive sampling at the producer, a very diverse L. monocytogenes population was found, with some strains persisting for several years. Listeria monocytogenes was found especially in niches of the food processing environment, difficult to reach for cleaning and disinfection. This study pointed to weaknesses in industrial hygiene that resulted in the production of contaminated food and thus triggered the listeriosis outbreak. If L. monocytogenes is found in a food product, the European RASFF enables international communication of this risk and, for example, immediate product recalls. In this way, it makes an essential contribution to protecting consumers from foodborne infections. An analysis of the RASFF notifications concerning L. monocytogenes as part of this doctoral project highlighted the complexity of international processing and distribution channels. This underlines how important international cooperation is with regard to food safety. The fact that individual producers were involved in many RASFF notifications in different years again showed the influence of inadequate industrial hygiene and persistence on the spread of L. monocytogenes. In addition, the results of the study highlighted the need for food processing companies to take greater responsibility for their own operations. The success of the WGS-based surveillance strategy for L. monocytogenes, which was established as part of this doctoral project and continuously optimised over its course, was already evident during the project term. For the first time, the number of reported cases of listeriosis in Germany no longer increased in 2018, but decreased, and continued to decrease in 2019. The dissertation project has thus made a decisive contribution to controlling the spread of L. monocytogenes along the food chain and thus reducing the number of listeriosis cases in Germany., Die humane Listeriose ist eine vergleichsweise seltene, aber schwerwiegende Infektionserkrankung. Die hohe Hospitalisierungs- und Sterblichkeitsrate machen sie weltweit zu einem großen Problem für die öffentliche Gesundheit. Ältere, Schwangere und Immungeschwächte haben ein erhöhtes Infektionsrisiko. Verursacht wird die Listeriose durch den Verzehr von Lebensmitteln, die mit dem Bakterium Listeria (L.) monocytogenes kontaminiert sind. Listeria monocytogenes ist weit verbreitet in der Umwelt und Tierwelt. In die Lebensmittelkette gelangt es entweder über kontaminierte rohe tierische oder pflanzliche Produkte oder über Kreuzkontaminationen während der Verarbeitung von Lebensmitteln. Das übergeordnete Ziel der vorliegenden Arbeit war es, mittels modernster molekularepidemiologischer Methoden und genetischem Profiling von Bakterienisolaten Listeriosefälle beim Menschen zu verhindern. Neben der klassischen Epidemiologie werden molekulare Typisierungsmethoden angewendet, um den Eintrag und die Verbreitung von L. monocytogenes entlang der Lebensmittelkette bis hin zum Verbraucher aufzudecken. In den letzten Jahren wurde die molekulare Typisierung durch die Möglichkeit zur Gesamtgenomsequenzierung (WGS, whole genome sequencing) bakterieller Isolate revolutioniert. Anhand der in der vorliegenden Promotionsarbeit gewonnenen Erkenntnisse konnte der bisherige Goldstandard zur Feintypisierung, die Pulsed-Field-Gelektrophorese, im Nationalen Referenzlabor (NRL) für L. monocytogenes in 2018 komplett durch die WGS abgelöst werden. Der Wert der WGS als hochauflösendes, molekulares Überwachungstool für L. monocytogenes wurde im Rahmen des Promotionsprojekts umfassend an die Stakeholder entlang der Lebensmittelkette kommuniziert. Auf der Basis der im Zuge des Promotionsprojekts im NRL etablierten Protokolle steht das Tool den nationalen Überwachungsbehörden nun zur Verfügung. Innerhalb des Promotionsvorhabens wurde die bioinformatische Analyse von WGS-Daten von L. monocytogenes für den Datenaustausch optimiert, beispielhaft ein Listerioseausbruch und die Verbreitung von Listeria spp. in einem lebensmittelverarbeitenden Betrieb untersucht, sowie die Bedeutung des europäischen Schnellwarnsystems für lebensmittelbedingte Krankheitsausbrüche evaluiert. Mittels WGS lassen sich Kontaminations- und Infektionsketten mit beispielloser Genauigkeit nachvollziehen und so unterbrechen. Die Verfahren zur Analyse von WGS-Daten sind jedoch vielfältig und noch nicht umfassend harmonisiert, was den Datenaustausch erschwert. In der vorliegenden Arbeit wurde gezeigt, dass mit verschiedenen WGS-Analysemethoden für L. monocytogenes größtenteils vergleichbare Ergebnisse generiert werden können. Ein innerhalb der Promotionsarbeit entwickelter Übersetzungscode ermöglicht es, Informationen zu Clustern auch ohne harmonisierte Methoden zwischen Sektoren (z.B. Lebensmittelsicherheit, Öffentliches Gesundheitswesen) und Ländern auszutauschen. Dieses Vorgehen bietet einen großen Vorteil, indem es gerade in zeitkritischen Situationen wie Listerioseausbrüchen eine schnelle Kommunikation zwischen den Stakeholdern erlaubt. Eine wichtige Voraussetzung, um Listerioseinfektionen zu verhindern, ist ein stärkerer Fokus auf die Betriebshygiene in der Lebensmittelverarbeitung. Deutlich wurde dies am Beispiel einer im Rahmen der Promotionsarbeit durchgeführten Studie einer Geflügelverarbeitungskette. Mittels WGS-Typisierung konnte gezeigt werden, dass Listeria spp. vom Tier und aus der Umwelt bis in das fertige Lebensmittelprodukt übertragen werden. Zusätzlich zeigte sich eine Ausbreitung auf Oberflächen in der Produktionsumgebung, wodurch gefährliche Kreuzkontaminationen begünstigt werden. Seit der Einführung der WGS für die Typisierung von L. monocytogenes in Deutschland konnte in sektorübergreifender Zusammenarbeit eine Vielzahl von Listerioseausbrüchen aufgeklärt und beendet werden. Die vorliegende Arbeit gibt Einblick in einen großen, über mehrere Jahre anhaltenden, nationalen Listeriseausbruch, der im Rahmen des Promotionsvorhabens federführend untersucht wurde. Mittels WGS-Typisierung konnte der Ausbruch auf verzehrfertige Fleischprodukte und deren Herstellerbetrieb zurückverfolgt werden. Durch eine vorwärts gerichtete Überprüfung (forward checking) als Ergänzung zu der in Ausbruchsuntersuchungen üblichen Rückverfolgung (backtracking), konnten zwei verschiedene Cluster von Listeriosefällen demselben Betrieb zugeordnet werden. Nur durch solch eine zweiseitige Kontrollstrategie, bei der backtracking und forward checking kombiniert werden, können Listerioseausbrüche in Zukunft wesentlich minimiert werden. Als Resultat der intensiven Beprobung des Herstellerbetriebs zeigte sich eine sehr diverse L. monocytogenes-Population mit teils über mehrere Jahre persistierenden Stämmen. Listeria monocytogenes wurde insbesondere in für Reinigung und Desinfektion schwer zu erreichenden Nischen gefunden. Diese Studie deutete auf Schwachpunkte in der Betriebshygiene hin, die die Produktion kontaminierter Lebensmittel nach sich zogen und so Auslöser des Listerioseausbruchs waren. Wird L. monocytogenes in einem Lebensmittel gefunden, ermöglicht das europäische Schnellwarnsystem für Lebensmittel und Futtermittel (RASFF, Rapid Alert System for Food and Feed) eine internationale Kommunikation dieses Risikos und beispielsweise umgehende Produktrückrufe. Auf diese Weise trägt es essenziell dazu bei, Verbraucher vor lebensmittelbedingten Infektionen zu schützen. Die Analyse von RASFF-Meldungen bezüglich L. monocytogenes im Rahmen der vorliegenden Arbeit verdeutlichte die Komplexität internationaler Verarbeitungs- und Vertriebswege. Dies unterstreicht, wie wichtig auch die internationale Zusammenarbeit im Hinblick auf die Lebensmittelsicherheit ist. Die Tatsache, dass einzelne lebensmittelverarbeitende Betriebe in zahlreiche RASFF-Meldungen in unterschiedlichen Jahren involviert waren, zeigte erneut den Einfluss unzureichender Betriebshygiene und Persistenz auf die Ausbreitung von L. monocytogenes. Zudem verdeutlichten die Studienergebnisse den Bedarf einer stärkeren Eigenverantwortung lebensmittelverarbeitender Betriebe. Schon während der Laufzeit zeigte sich der Erfolg der im Rahmen des Promotionsprojekts etablierten und im Projektverlauf stetig optimierten WGS-basierten Überwachungsstrategie von L. monocytogenes. Die Zahl gemeldeter Listeriosefälle in Deutschland war in 2018 erstmals nicht mehr ansteigend, sondern rückläufig, und sank in 2019 weiter. Das Promotionsvorhaben hat damit entscheidend dazu beigetragen, die Ausbreitung von L. monocytogenes entlang der Nahrungskette zu kontrollieren und so die Zahl der Listerioseerkrankungen in Deutschland zu reduzieren.

El incremento de las temperaturas regionales y la intensidad de uso de las tierras en el Escudo de Guayana y la Amazonia est��n imponiendo una reducci��n acelerada de las poblaciones de especies m��s sensibles, por lo que es urgente realizar inventarios biol��gicos en las zonas m��s vulnerables. Con la finalidad de conocer la diversidad y distribuci��n de l��quenes en la regi��n del Alto Orinoco, Parque Nacional Duida-Marahuaca y sus ��reas adyacentes en el estado Amazonas, Venezuela, se realiz�� un inventario principalmente a partir de cuatro expediciones donde se colectaron espec��menes en m��s de 40 sitios en pisos altitudinales ubicados entre los 200 m (bosque basimontano y sabanas) y por encima de 1500 m (vegetaci��n altotepuyana). Datos adicionales se obtuvieron a partir de la literatura y herbarios. Aunque los resultados no pretenden ser completos, revelaron un total de 205 especies descritas, 150 especies indeterminadas, 84 g��neros y 27 familias. Entre las especies descritas, se encuentran 162 presentes en la franja basimontana, 38 en la franja montana, 24 en la franja altotepuyana y 20 end��micas. Se reportan 69 nuevos registros procedentes estrictamente de las distintas franjas altitudinales del Cerro Duida. Se presenta una lista revisada con datos taxon��micos y ecol��gicos. Se describen como nuevas cinco especies y una variedad: Cladonia duidana V.Marcano & A.Morales sp. nov. (Cladoniaceae), Pertusaria orinoquensis V.Marcano sp. nov., Sticta kunuhana V.Marcano sp. nov. (Lobariaceae), S. spruceana V.Marcano sp. nov. (Lobariaceae), Xanthoparmelia esmeraldensis V.Marcano & A.Morales sp. nov. (Parmeliaceae) y Lepraria arbuscula (Nyl.) Lendemer & Hodk. var. fumarprotocetrarica V.Marcano var. nov., Increasing temperature and changing land-use in the Guayana Shield and Amazonia result in an accelerated decline of sensitive lichen populations. Monitoring of these populations by biological plot inventories in particularly vulnerable sites is urgently needed. In order to know the diversity and distribution of lichen species at the Alto Orinoco, Parque Nacional Duida-Marahuaca and nearby areas from Amazonas state, Venezuela, lichens were sampled during four expeditions in more than 40 plots located from 200 m (premontane forest) to more than 1500 m elevation (altotepuyana vegetation). Additional data were obtained from literature and herbaria. Our assessment, although incomplete, revealed 205 described species, 150 undescribed species, 84 genera and 27 families. Among the described species 162 were observed in the basimontane vegetation, 38 in the montane vegetation, 24 in the altotepuyana vegetation, while 20 appear to be endemic to the study area. Sixty-nine species are new records for the Cerro Duida. A checklist with taxonomic and ecological data is presented. Five new species and one new variety are described: Cladonia duidana V.Marcano & A.Morales sp. nov. (Cladoniaceae), Pertusaria orinoquensis V.Marcano sp. nov., Sticta kunuhana V.Marcano sp. nov. (Lobariaceae), S. spruceana V.Marcano sp. nov. (Lobariaceae), Xanthoparmelia esmeraldensis V.Marcano & A.Morales sp. nov. (Parmeliaceae), and Lepraria arbuscula (Nyl.) Lendemer & Hodk. var. fumarprotocetrarica V.Marcano var. nov.

The taxonomy of the genus Sticta in Hawaii is reassessed, based on a separately published molecular phylogeny using the fungal barcoding marker ITS. Based on Magnusson and Zahlbruckner's treatment from 1943 and Magnusson's catalogue from 1955, seven species of Sticta and three infraspecific taxa had been reported from the archipelago, all widespread except the putative endemic S. plumbicolor. Here we provide a taxonomic treatment of 13 taxa, 12 species and one subspecies, distinguished in a previous phylogenetic analysis: S. acyphellata, S. andina, S. antoniana, S. emmanueliana, S. flynnii, S. fuliginosa, S. hawaiiensis, S. limbata, S. plumbicolor, S. scabrosa subsp. hawaiiensis, S. smithii, S. tomentosa and S. waikamoi. All taxa are described, discussed and illustrated and a dichotomous key is presented. The implications of revised species taxonomies for studies in other fields such as ecology, ecophysiology, biogeography, biochemistry, and applications such as environmental monitoring are discussed. We also propose a protocol to use Sticta lichens to monitor the environmental health of Hawaiian ecosystems.

DNA barcoding using the nuclear internal transcribed spacer (ITS) has become prevalent in surveys of fungal diversity. This approach is, however, associated with numerous caveats, including the desire for speed, rather than accuracy, through the use of automated analytical pipelines, and the shortcomings of reference sequence repositories. Here we use the case of a specimen of the bracket fungus Trametes s.lat. (which includes the common and widespread turkey tail, T. versicolor) to illustrate these problems. The material was collected in Vietnam as part of a biodiversity inventory including DNA barcoding approaches for arthropods, plants and fungi. The ITS barcoding sequence of the query taxon was compared against reference sequences in GenBank and the curated fungal ITS database UNITE, using BLASTn and MegaBLAST, and was subsequently analysed in a multiple alignment-based phylogenetic context through a maximum likelihood tree including related sequences. Our results initially indicated issues with BLAST searches, including the use of pairwise local alignments and sorting through Total score and E value, rather than Percentage identity, as major shortcomings of the DNA barcoding approach. However, after thorough analysis of the results, we concluded that the single most important problem of this approach was incorrect sequence labelling, calling for the implementation of third-party annotations or analogous approaches in primary sequence repositories. In addition, this particular example revealed problems of improper fungal nomenclature, which required reinstatement of the genus name Cubamyces (= Leiotrametes), with three new combinations: C. flavidus, C. lactineus and C. menziesii. The latter was revealed as the correct identification of the query taxon, although the name did not appear among the best BLAST hits. While the best BLAST hits did correspond to the target taxon in terms of sequence data, their label names were misleading or unresolved, including [Fungal endophyte], [Uncultured fungus], Basidiomycota, Trametes cf. cubensis, Lenzites elegans and Geotrichum candidum (an unrelated ascomycetous contaminant). Our study demonstrates that accurate identification of fungi through molecular barcoding is currently not a fast-track approach that can be achieved through automated pipelines.Citation: Lucking R., Truong B. V., Huong D. T. T., Le N. H., Nguyen Q. D., Nguyen V. D., Raab-Straube E. von, Bollendorff S., Govers K. & Di Vincenzo V. 2020: Caveats of fungal barcoding: a case study in Trametes s.lat. (Basidiomycota: Polyporales) in Vietnam reveals multiple issues with mislabelled reference sequences and calls for third-party annotations. – Willdenowia 50: 383–403. doi: https://doi.org/10.3372/wi.50.50302Version of record first published online on 15 September 2020 ahead of inclusion in December 2020 issue.

Strigulasensu lato has been previously defined based on phenotype characters as a rather broad genus including tropical to temperate species growing on a wide array of substrata. In this study, based on a multilocus phylogenetic approach, we show that foliicolous species form six well-delimited clades that correlate with diagnostic phenotype features, including thallus morphology, carbonization of the involucrellum and excipulum, ascospore dimensions, and type of macroconidia. Given the topology, with five of the six clades emerging on long stem branches, and the strong phenotypical differentiation between the clades, we recognize these at the genus level, making mostly use of previously established genus names. Four genera, namelyPhylloporis,Puiggariella,Raciborskiella, andRacoplaca, are resurrected for theS. phyllogena,S. nemathora,S. janeirensis, andS. subtilissimagroups, respectively, whereas one new genus,Serusiauxiellagen. nov., is introduced for a novel lineage with peculiar macroconidia. The only sequenced non-foliicolous species,S. jamesii, is not closely related to these six foliicolous lineages but clusters withFlavobatheliumandPhyllobathelium, revealing it as an additional undescribed genus-level lineage being treated elsewhere. Within the new genusSerusiauxiella, three new species are described:Serusiauxiella filiferasp. nov.,S. flagellatasp. nov., andS. sinensissp. nov. In addition, ten new combinations are proposed:Phylloporis austropunctatacomb. nov.,P. radiatacomb. nov.,P. vulgariscomb. nov.,Puiggariella confluenscomb. et stat. nov.,P. nemathoracomb. nov.,P. nigrocinctacomb. nov.,Racoplaca maculatacomb. nov.,R. melanobaphacomb. nov.,R. transversoundulata, andR. tremenscomb. nov. We also report on a peculiar, previously unrecognized growth behaviour of the macroconidial appendages inStrigulas.lat.