Ellen L. Zechner's research while affiliated with Karl-Franzens-Universität Graz and other places

Background Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. Methods We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. Results In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. Conclusion H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.

Objectives: In infants with suspected food protein induced proctocolitis (sFPIP) only a minority of patients are finally diagnosed with the disease following diagnostic dietary intervention (DDI). There is a need for a pathophysiological explanation for the cause of hematochezia in the majority of sFPIP infants. Methods: We prospectively recruited infants with sFPIP and healthy controls. Fecal samples were collected at inclusion, week 4 (end of DDI in sFPIP) and week 8. For 16S rRNA sequencing (515F/806R) we used Illumina MiSeq. Amplicon sequence variants were generated using Qiime2 and DADA2. Qiime diversity alpha and beta group comparisons and linear discriminant analysis effect size analysis was performed. For shotgun metagenomic analysis on species level we used KneadData and MetaPhlAn2. Results: 14 sFPIP infants were compared to 55 healthy infants. At inclusion overall microbial composition of sFPIP infants differed significantly from controls (weighted UniFrac;PairwisePERMANOVA, p=0.002, pseudo-F=5.008). On genus level healthy infant microbiota was significantly enriched with Bifidobacterium (B.) compared to sFPIP patients (LDA=5.5,p<0.001,31.3vs12.1%). sFPIP stool was significantly enriched by Clostridium sensu stricto 1 over controls (LDA=5.3,p=0.003,3.5vs18.3%). DDI caused a significant and sustained increase of Bifidobacterium (LDA=5.4,p=0.048,27.9%) in sFPIP infants. Species level analysis revealed significant reduction of abundance of B. longum in sFPIP patients, which after DDI was reversed by B. species other than B. longum. Conclusions: We revealed a gut microbiota dysbiosis phenomenon in sFPIP infants. DDI induces a microbiota composition comparable to that of healthy infants. In most sFPIP infants hematochezia might be triggered by a gut microbiota dysbiosis phenomenon.

Klebsiella oxytoca is a ubiquitous bacterium that is increasingly associated with inflammatory diseases. Here, we report the hybrid assembled genome for cytotoxic K. oxytoca strain AHC-6. The genome comprises a total of 5.7 Mbp, with a GC content of 55.2% and 5,258 coding sequences after assembly and annotation.

The DNA-alkylating metabolite tilimycin is a microbial genotoxin. Intestinal accumulation of tilimycin in individuals carrying til+ Klebsiella spp. causes apoptotic erosion of the epithelium and colitis. Renewal of the intestinal lining and response to injury requires the activities of stem cells located at the base of intestinal crypts. This study interrogates the consequences of tilimycin-induced DNA damage to cycling stem cells. We charted the spatial distribution and luminal quantities of til metabolites in Klebsiella-colonized mice in the context of a complex microbial community. Loss of marker gene G6pd function indicates genetic aberrations in colorectal stem cells that became stabilized in monoclonal mutant crypts. Mice colonized with tilimycin-producing Klebsiella displayed both higher frequencies of somatic mutation and more mutations per affected individual than animals carrying a non-producing mutant. Our findings imply that genotoxic til+ Klebsiella may drive somatic genetic change in the colon and increase disease susceptibility in human hosts.

[This corrects the article DOI: 10.3389/fmicb.2021.692453.].

Klebsiella spp. that secrete the DNA-alkylating enterotoxin tilimycin colonize the human intestinal tract. Numbers of toxigenic bacteria increase during antibiotic use, and the resulting accumulation of tilimycin in the intestinal lumen damages the epithelium via genetic instability and apoptosis. Here we examine the impact of this genotoxin on the gut ecosystem. 16S rRNA sequencing of faecal samples from mice colonized with Klebsiella oxytoca strains and mechanistic analyses show that tilimycin is a pro-mutagenic antibiotic affecting multiple phyla. Transient synthesis of tilimycin in the murine gut antagonized niche competitors, reduced microbial richness and altered taxonomic composition of the microbiota both during and following exposure. Moreover, tilimycin secretion increased rates of mutagenesis in co-resident opportunistic pathogens such as Klebsiella pneumoniae and Escherichia coli, as shown by de novo acquisition of antibiotic resistance. We conclude that tilimycin is a bacterial mutagen, and flares of genotoxic Klebsiella have the potential to drive the emergence of resistance, destabilize the gut microbiota and shape its evolutionary trajectory. Production of the enterotoxin tilimycin by gut-resident Klebsiella species can alter gut microbiota composition, induce mutational evolution and drive the emergence of antibiotic resistance in mice.

Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transconjugant and donor cells. The development of robust, fast and reliable techniques for in situ monitoring and quantification of conjugation ratios might accelerate progress in understanding the impact of this cellular process in the HGT. The IncHI1 plasmids, involved in multiresistance phenotypes of relevant pathogens such as Salmonella and E. coli, are distinguished by the thermosensitivity of their conjugative transfer. Conjugation mediated by IncHI1 plasmids is more efficient at temperatures lower than 30 °C, suggesting that the transfer process takes place during the environmental transit of the bacteria. In this report, we described a methodology to monitor in situ the conjugation process during agar surface matings of the IncHI1 plasmid R27 and its derepressed derivative drR27 at different temperatures. A three-color-labeling strategy was used to visualize the spatial distribution of transconjugants within the heterogeneous environment by epifluorescence and confocal microscopy. Moreover, the fluorescent labelling was also used to quantify conjugation frequencies in liquid media by flow cytometry.

The human gut harbors a complex community of microbes, including several species and strains that could be commensals or pathogens depending on context. The specific environmental conditions under which a resident microbe changes its relationship with a host and adopts pathogenic behaviors, in many cases, remain poorly understood.

Objectives: Klebsiella (K.) oxytoca is a gastrointestinal pathobiont with the potential to produce the toxins tilivalline and tilimycin which cause antibiotic-associated hemorrhagic colitis. Overgrowth of toxigenic K. oxytoca has recently been implicated in necrotizing enterocolitis. K. oxytoca colonises 2-9% of healthy adults, however, there is no systematic data on colonisation in healthy children. We investigated K. oxytoca colonisation and its toxigenic properties in healthy infants. Methods: We sampled stool of healthy infants and determined K. oxytoca colonisation using stool culture and PCR (pehX). Toxin in stool was measured with HPLC/high-resolution mass spectrometry. K. oxytoca isolates were typed using MLST and K. oxytoca toxin PCR (npsA/B). Cytotoxin production of isolates was analysed by MTT assay. Results: K. oxytoca was detected in 30 /61 infants (49%) using stool culture and in 45/61 (73%) using PCR (pehX). Toxin marker PCR (npsA/B) was positive in 66% of stool samples positive for K. oxytoca PCR. Stool toxin levels were too low for quantitation but traces of tilivalline were detected. Contrarily, 49% of K. oxytoca isolates demonstrated toxicity in the MTT assay. MLST revealed 36 distinct sequence types affiliated with all known K. oxytoca sequence type clusters (A, B1 and B2). Conclusions: More than 70% of healthy infants were colonized with K. oxytoca. Toxin quantities in stool of colonized healthy infants were below detection level, yet half of the isolates produced toxin in vitro demonstrating their pathobiont potential. The high occurrence of toxigenic K. oxytoca in healthy infants has to be considered for future disease association studies.

Members of the Klebsiella oxytoca species complex (KoSC) are emerging human pathogens causing infections of increasing significance especially in healthcare settings. KoSC strains are affiliated with distinct phylogroups based on genetic variation at the beta-lactamase gene (blaOXY) and it has been proposed that each major phylogroup represents a unique species. However, since the typing methods applied in clinical settings cannot differentiate every species within the complex, existing clinical, epidemiological and DNA sequence data is frequently misclassified. Here we systematically examined the phylogenetic relationship of KoSC strains to evaluate robustness of existing typing methods and to provide a simple typing strategy for KoSC members that cannot be differentiated biochemically. Initial analysis of a collection of K. oxytoca, K. michiganensis, K. pasteurii, and K. grimontii strains of environmental origin showed robust correlation of core phylogeny and blaOXY grouping. Moreover, we identified species-specific accessory gene loci for these strains. Extension of species correlation using database entries initially failed. However, assessment of average nucleotide identities (ANI) and phylogenetic validations showed that nearly one third of isolates in public databases have been misidentified. Reclassification resulted in a robust reference strain set for reliable species identification of new isolates or for retyping of strains previously analyzed by multi-locus sequence typing (MLST). Finally, we show convergence of ANI, core gene phylogeny, and accessory gene content for available KoSC genomes. We conclude that also the monophyletic members K. oxytoca, K. michiganensis, K. pasteurii and K. grimontii can be simply differentiated by a PCR strategy targeting blaOXY and accessory genes defined here.