An adapted method for Cas9-mediated editing reveals the species-specific role of ß-glucoside utilization driving competition between Klebsiella species.

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. Firstly, the system allowed efficient and precise full-length gene deletion via enhanced lambda Red expression. Furthermore, we tested the efficiency of two independent, functionally validated complementation strategies. Ultimately, the insertion of universal "bookmark" targets during gene deletion subsequently allows the most optimal genetic complementation in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. This approach offers a significant advantage by enabling the use of a single high-efficiency "bookmark" for complementing other loci or strains, eliminating the need for site-specific design. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. IMPORTANCE: Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We then adapted a recently developed protocol for functional complementation based on universal "bookmark" targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.

Reduced intestinal lipid absorption improves glucose metabolism in aged G2-Terc knockout mice.

BACKGROUND: Biological aging is an important factor leading to the development of pathologies associated with metabolic dysregulation, including type 2 diabetes, cancer, cardiovascular and neurodegenerative diseases. Telomere length, a central feature of aging, has additionally been identified as inversely associated with glucose tolerance and the development of type 2 diabetes. However, the effects of shortened telomeres on body weight and metabolism remain incompletely understood. Here, we studied the metabolic consequences of moderate telomere shortening using second generation loss of telomerase activity in mice. RESULTS: Aged male and female G2 Terc-/- mice and controls were characterized with respect to body weight and composition, glucose homeostasis, insulin sensitivity and metabolic activity. This was complemented with molecular and histological analysis of adipose tissue, liver and the intestine as well as microbiota analysis. We show that moderate telomere shortening leads to improved insulin sensitivity and glucose tolerance in aged male and female G2 Terc-/- mice. This is accompanied by reduced fat and lean mass in both sexes. Mechanistically, the metabolic improvement results from reduced dietary lipid uptake in the intestine, characterized by reduced gene expression of fatty acid transporters in enterocytes of the small intestine. Furthermore, G2-Terc-/- mice showed significant alterations in the composition of gut microbiota, potentially contributing to the improved glucose metabolism. CONCLUSIONS: Our study shows that moderate telomere shortening reduces intestinal lipid absorption, resulting in reduced adiposity and improved glucose metabolism in aged mice. These findings will guide future murine and human aging studies and provide important insights into the age associated development of type 2 diabetes and metabolic syndrome.

Colitis ameliorates cholestatic liver disease via suppression of bile acid synthesis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of the biliary tree. The majority of PSC patients suffer from concomitant inflammatory bowel disease (IBD), which has been suggested to promote disease development and progression. However, the molecular mechanisms by which intestinal inflammation may aggravate cholestatic liver disease remain incompletely understood. Here, we employ an IBD-PSC mouse model to investigate the impact of colitis on bile acid metabolism and cholestatic liver injury. Unexpectedly, intestinal inflammation and barrier impairment improve acute cholestatic liver injury and result in reduced liver fibrosis in a chronic colitis model. This phenotype is independent of colitis-induced alterations of microbial bile acid metabolism but mediated via hepatocellular NF-κB activation by lipopolysaccharide (LPS), which suppresses bile acid metabolism in-vitro and in-vivo. This study identifies a colitis-triggered protective circuit suppressing cholestatic liver disease and encourages multi-organ treatment strategies for PSC.

Microbiota of vaccinated and non-vaccinated clinically inconspicuous and conspicuous piglets under natural Lawsonia intracellularis infection.

Lawsonia (L.) intracellularis is a widespread, economically important bacterium causing the porcine proliferative enteropathy (PPE). In this study, we evaluated intestinal microbiota of naturally exposed L. intracellularis-positive pigs under standardized conditions. To obtain three independent repetitions, 27 L. intracellularis-infected pigs (19.0 ± 1.50 kg body weight) from one farm were divided into three groups at an age of 7 to 8 weeks (nine pigs/group). Pigs were either vaccinated against L. intracellularis via oral drenching on their 21st day of life (attenuated live vaccine) or non-vaccinated and selected according to clinical findings (pigs without deviating fecal consistency or with moderate to soft fecal consistency). Comparison of the clinically inconspicuous piglets that differed regarding their vaccination status showed fewer significant differences in fecal microbiota composition. The vaccination led to an overall enrichment of bacterial species belonging to the order Clostridiales, while species of the genus Collinsella and Prevotella were decreased. Several bacterial species belonging to the order Bacteroidales, mainly of the family Prevotellacecae, often closely matching Prevotella copri differed significantly between non-vaccinated clinically inconspicuous and conspicuous piglets. Whether those bacterial species play a role in mitigating the severity of an L. intracellularis infection remains to be defined.

High Dietary Intake of Rye Affects Porcine Gut Microbiota in a Salmonella Typhimurium Infection Study.

Bacterial fermentation of undigested carbohydrates in the hindgut has considerable potential for the stimulation or inhibition of the growth of distinct bacteria within microbiota. The aim of the present study was to evaluate whether high levels of rye affect porcine gut microbiota composition with subsequent effects on the load of Salmonella Typhimurium, an intestinal pathogen with zoonotic relevance. Therefore, forty-two 25-day-old piglets were allocated to two groups and fed a diet containing either 69% wheat or 69% rye for 35 days. One week after introducing the two different diets, the piglets were experimentally infected with Salmonella Typhimurium. The microbiota composition of cecal and fecal samples of the piglets were evaluated 28 days after infection. In the cecum, promoted growth of Bifidobacterium, several lactic acid bacteria and Faecalibacterium prausnitzii were seen in pigs fed the diet containing 69% rye. Bacterial species belonging to the genera Bifidobacterium and Catenisphaera were associated with differing bacterial counts of Salmonella Typhimurium detected in the cecal contents of all piglets in both feeding groups via cultural cultivation. The high intake of rye instead of wheat seems to promote the growth of beneficial intestinal bacteria accompanied by impaired growth conditions for the foodborne pathogen Salmonella Typhimurium.

Imbalanced gut microbiota fuels hepatocellular carcinoma development by shaping the hepatic inflammatory microenvironment.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and therapeutic options for advanced HCC are limited. Here, we observe that intestinal dysbiosis affects antitumor immune surveillance and drives liver disease progression towards cancer. Dysbiotic microbiota, as seen in Nlrp6-/- mice, induces a Toll-like receptor 4 dependent expansion of hepatic monocytic myeloid-derived suppressor cells (mMDSC) and suppression of T-cell abundance. This phenotype is transmissible via fecal microbiota transfer and reversible upon antibiotic treatment, pointing to the high plasticity of the tumor microenvironment. While loss of Akkermansia muciniphila correlates with mMDSC abundance, its reintroduction restores intestinal barrier function and strongly reduces liver inflammation and fibrosis. Cirrhosis patients display increased bacterial abundance in hepatic tissue, which induces pronounced transcriptional changes, including activation of fibro-inflammatory pathways as well as circuits mediating cancer immunosuppression. This study demonstrates that gut microbiota closely shapes the hepatic inflammatory microenvironment opening approaches for cancer prevention and therapy.

Klebsiella oxytoca causes colonization resistance against multidrug-resistant K. pneumoniae in the gut via cooperative carbohydrate competition.

Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

Removable denture is a risk indicator for peri-implantitis and facilitates expansion of specific periodontopathogens: a cross-sectional study.

BACKGROUND: The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis. METHODS: During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified. RESULTS: 16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (p = < 0.001) and implant level (p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present. CONCLUSIONS: Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation.

Single-Nucleotide Polymorphism-Based Genetic Diversity Analysis of Clinical Pseudomonas aeruginosa Isolates.

Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Because different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here, we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single-nucleotide polymorphisms that are the basis for branching of the phylogenetic tree. We also identified single-nucleotide polymorphisms that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. Although our results demonstrate that the Pseudomonas aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.

BACTOME-a reference database to explore the sequence- and gene expression-variation landscape of Pseudomonas aeruginosa clinical isolates.

Extensive use of next-generation sequencing (NGS) for pathogen profiling has the potential to transform our understanding of how genomic plasticity contributes to phenotypic versatility. However, the storage of large amounts of NGS data and visualization tools need to evolve to offer the scientific community fast and convenient access to these data. We introduce BACTOME as a database system that links aligned DNA- and RNA-sequencing reads of clinical Pseudomonas aeruginosa isolates with clinically relevant pathogen phenotypes. The database allows data extraction for any single isolate, gene or phenotype as well as data filtering and phenotypic grouping for specific research questions. With the integration of statistical tools we illustrate the usefulness of a relational database structure for the identification of phenotype-genotype correlations as an essential part of the discovery pipeline in genomic research. Furthermore, the database provides a compilation of DNA sequences and gene expression values of a plethora of clinical isolates to give a consensus DNA sequence and consensus gene expression signature. Deviations from the consensus thereby describe the genomic landscape and the transcriptional plasticity of the species P. aeruginosa. The database is available at https://bactome.helmholtz-hzi.de.

Deep transcriptome profiling of clinical Klebsiella pneumoniae isolates reveals strain and sequence type-specific adaptation.

Health-care-associated infections by multi-drug-resistant bacteria constitute one of the greatest challenges to modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds, among which the acquisition of carbapenemases is one of the most concerning. In Klebsiella pneumoniae, the dissemination of the K. pneumoniae carbapenemase is tightly connected to the global spread of certain clonal lineages. Although antibiotic resistance is a key driver for the global distribution of epidemic high-risk clones, there seem to be other adaptive traits that may explain their success. Here, we exploited the power of deep transcriptome profiling (RNA-seq) to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origins. We identified a large set of 3346 genes which was expressed in all isolates. While the core-transcriptome profiles varied substantially between groups of different sequence types, they were more homogenous among isolates of the same sequence type. We furthermore linked the detailed information on differentially expressed genes with the clinically relevant phenotypes of biofilm formation and bacterial virulence. This allowed for the identification of a diminished expression of biofilm-specific genes within the low biofilm producing ST258 isolates as a sequence type-specific trait.

In vivo mRNA profiling of uropathogenic Escherichia coli from diverse phylogroups reveals common and group-specific gene expression profiles.

mRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression of Escherichia coli pathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associated E. coli isolates to different phylogenetic groups. Whereas the in vivo gene expression profiles of the majority of genes were conserved among 21 E. coli strains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribed in vivo relative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease. Importance: Urinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenic Escherichia coli strains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenic E. coli gene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.