7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof.Dr.

Chair

Prof. Dr.

Professor

A culture-free whole-genome sequencing approach for rapid detection and characterization of Helicobacter pylori in gastric biopsies

Gastrointestinal Infections

Helicobacter pylori is a gastric pathogen with a high worldwide prevalence and a significant clinical burden. Chronic infection leads to chronic gastritis and can progress to other disorders such as ulcers, MALT lymphoma or gastric adenocarcinoma. The emergence of multi-resistant strains is responsible for increasing failure rates of first-line combination therapies and the WHO classified H. pylori as a high priority organism for the development of novel antibiotics in 2017. Current clinical guidelines for the eradication of H. pylori typically recommend antibiotic susceptibility testing only after unsuccessful first-line treatments. However, culture-based susceptibility testing of H. pylori can take up to two weeks and thus empirical drug therapies are generally started before results are available.To address this clinical situation, we developed a microbial enrichment pipeline allowing us to sequence whole H. pylori genomes from DNA directly extracted from gastric biopsies obtained during routine endoscopy. The pipeline comprises multiple rounds of enrichment with native genomic DNA, sequencing libraries and real-time Nanopore sequencing. Using this approach, we were able to obtain complete H. pylori genomes with 5-10x coverage, representing a 50 to 100-fold enrichment of the low-abundance bacterial DNA within gastric tissue samples. The resulting genomic data can then be used for further characterization relevant to clinical diagnosis or public health surveillance. In particular, we performed a genotype-based analysis to predict susceptibility to clinically relevant antibiotics and obtained accuracies ranging from 93% (clarithromycin) to 62% (metronidazole).In summary, our approach allows for quick determination of antibiotic susceptibility in H. pylori which can ultimately be implemented as a rapid diagnostic test to optimize therapeutic efficiency and curtail the development of antibiotic resistance in this pathogen.

Oral Presentation

Ingestion of sugar substitutes promotes gut colonization and virulence in salmonella typhimurium

Consumed by millions of people to manage blood glucose and body weight, sugar substitutes may have unintended side effects. Here, we found that the ingestion of arabinose, lactulose, maltitol, erythritol, and sorbitol, which can be substituted for table sugar, reduces colonization resistance against the common food borne pathogen, Salmonella Typhimurium and promotes its virulence. In mice, consumption of these sugars upon infection led to gut colonization by the pathogen characterized by high fecal shedding, a disseminated systemic infection and enteropathy. Although the gut microbiota can utilize these sugars, it is perturbed by intestinal acidification resulting from accumulating microbiota-derived fermentation byproducts, subsequently permitting pathogen colonization. In addition, Salmonella virulence gene expression is induced, which further enhances infection and intestinal disease. Moreover, the gut environment altered by these sugar substitutes selects for fully virulent, invasive Salmonella, as avirulent mutants are highly attenuated for growth in the gut. Interestingly, the diet can alter the pathogen's tissue tropism along the intestinal tract. These findings highlight that sugar substitutes in diets may pose a risk for enteric infections and offer a new model for studying how diet influences pathogen behavior and disease outcomes.

The natural product chlorotonil A spares the microbiota, preserves colonization resistance and prevents relapsing Clostridioides difficile infection

Clostridioides difficile infections (CDI) remain a major healthcare problem due to high rates of recurrent infections (rCDI). The treatment with broad-spectrum antibiotics often contributes to (r)CDI by sustained damage of the microbiota-mediated colonization resistance. Thus, the development of novel antibiotic candidates with distinct antimicrobial activity profiles against (r)CDI is of high public health interest to combat C. difficile infections.Within the frame of InfectControl 2020, we investigated the antimicrobial effects of a natural product compound, chlorotonil A (ChA) on C. difficile1. The compound in vitro showed bactericidal activity at concentrations comparable to those of standard antibiotics on a C. difficile strains. In a pilot ChA feeding experiment with pigs, we observed only minor effects on the overall composition of the gut microbiota, whereas the absolute abundance of the genera Terrisporobacter and Clostridium was significantly reduced. A transcriptome analysis of C. difficile revealed the disturbance and re-programming of key cellular functions even at sub-MIC concentrations of ChA.The notable microbiota-sparing property of ChA was confirmed in non-infected mice. In a CDI mouse model, ChA strikingly outperformed vancomycin in protecting mice against relapsing CDI. Subsequent experiments to characterize differences in anti-microbial activity revealed that ChA in contrast to vancomycin results in faster clearance of C. difficile vegetative cells and spores from the gut, a finding correlates with the faster recovery of the microbiota of ChA-treated C. difficile-infected mice. We also noted an unexpected direct activity of chlorotonils towards C. difficile spores, preventing their germination even after the removal of the antibiotic from the media. Together, chlorotonils act at similar concentrations on both vegetative cells and spores, defining a unique antimicrobial profile compared to existing antibiotics. Chlorotonils constitute a highly interesting compound family for the development as novel antibacterial agents to break the vicious cycle of rCDI.1Bublitz et al. (2023). Cell Host &amp; Microbe. doi: 10.1016/j.chom.2023.04.003.

SARS-CoV-2 tropism to intestine but not gastric epithelial cells is defined by limited ACE2 expression

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) infection primarily affects the lung but can also cause gastrointestinal symptoms. In vitro experiments confirmed that SARS-CoV-2 robustly infects intestinal epithelium. However, data on infection of adult gastric epithelium is sparse and a side-by-side comparison of the infection in the major segments of the gastrointestinal tract is lacking. We provide this direct comparison in organoid-derived monolayers and demonstrate that SARS-CoV-2 robustly infects intestinal epithelium, while gastric epithelium is resistant to infection. RNA sequencing and proteome analysis pointed to ACE2 as critical factor, and indeed, ectopic expression of ACE2 increased susceptibility of gastric organoid-derived monolayers to SARS-CoV-2. ACE2 expression pattern in patient gastrointestinal biopsies mirror SARS-CoV-2 infection levels in monolayers. Thus, local ACE2 expression limits SARS-CoV-2 expression in the GI tract to the intestine, suggesting that the intestine, but not the stomach is likely to be important in viral replication and possibly transmission.

Role of the CFTR in pathogenesis of Giardia duodenalis infections

IntroductionAs a widespread protozoal parasite, Giardia duodenalis is a common cause for malabsorption and diarrhea. Intestinal epithelial barrier defects are frequently observed in patients affected by the multifactorial disease giardiasis. While the exact pathomechanisms remain unknown, recent findings suggest a novel chain of events amounting in epithelial barrier breakdown, which starts with a disturbed ion homeostasis during the early infection phase. Amongst intestinal ion transporters, the anion channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) has been shown to be severely affected by Giardia, i.e. resulting in reduced protein expression and function.GoalsWe aim to shed light on the putative role of the CFTR channel in G. duodenalis infections by investigating its impact on pathogenesis at different infection stages.Materials &amp; MethodsUsing CRISPR/Cas9-technology, intestinal organoids from a healthy, CFTR wildtype donor were converted into CFTR-impaired CF-mimicking mutants. For comparison of CFTR-mutant and wildtype, the respective isogenic organoids were cultured in a compartmentalized transwell system, infected with G. duodenalis and the host response was analyzed at transcriptome level using single-cell RNA-sequencing (scRNAseq).ResultsscRNAseq experiments confirm a robust transcriptional downregulation of the CFTR ion transporter upon infection. Transcriptomic changes in intestinal organoids hint towards a severely altered host lipid metabolism and mitochondrial dysfunction in the late infection stage, which is significantly more pronounced in CFTR-impaired organoids.SummaryDue to the significant downregulation of the CFTR ion channel during G. duodenalis infections and its pathogenic role in a variety of different diseases, we hypothesized that CFTR non-functionality may play a greater role in giardiasis pathogenesis. Experiments using CFTR-impaired organoids indeed suggest an increased susceptibility to lipid imbalances and mitochondrial dysfunction downstream of functional CFTR impairment.

Raum 10-11

FG Gastrointestinale Infektionen & FG Eukaryontische Krankheitserreger & FG Mikrobielle Pathogenität & FG FG Mikrobiom (FG GI / FG EK / FG MP / FG Mikrobiom)

Oral Session
Pathogenesis of Gastrointestinal Infections - Mechanisms and Models

Appointment

Chair

Topic

Programme

Oral SessionPathogenesis of Gastrointestinal Infections - Mechanisms and Models

Appointment

Chair

Topic

Programme

Oral Session
Pathogenesis of Gastrointestinal Infections - Mechanisms and Models