Helmholtz Centre for Infection Research

Integrated genomic surveillance, combining whole genome sequencing (WGS) of bacterial isolates with patient movement data, promises improved detection and prevention of pathogen transmission. However, evidence on its cost-effectiveness and clinical utility remains limited, not least because the full extent of transmission in hospital settings is difficult to capture. We conducted a 28-month observational study at Rigshospitalet, Copenhagen, collecting patient movement data and sequencing 18 438 bacterial isolates from 7398 patients across diverse species, infection sites, and resistance profiles. We estimated the hypothetical benefits of implementing integrative WGS surveillance, assuming that continued transmission could be prevented when WGS information was acted upon immediately. We found that 1975 of 7398 of culture-positive hospitalized patients (26.7%) harboured a pathogen genetically related to another patient's isolate. Of those, 1359 (68.8%) had an epidemiological link in the hospital, with Enterococcus faecium by far being the most prevalent bacteria involved in transmissions. WGS-informed prevention could hypothetically generate a net savings of €1.35 million annually if transmission was stopped once a clonal isolate was detected in a second patient. Furthermore, this approach could potentially have prevented an estimated 1284 hospital-acquired infections over the 28-month study period, including 94 cases characterized by the recovery of bloodstream isolates. Our integrated genomic surveillance approach reveals previously unexplored transmission landscapes. We discovered that transmission is widespread, varies between species, and is not limited to resistant isolates. Our results emphasize the potential of integrated genomic surveillance, the identification of local transmission hotspots, potential for greater savings by including susceptible isolates, and an incremental cost-effectiveness ratio classification by pathogen species.

Abstract To protect themselves against bacteriophage infection, bacteria encode a vast diversity of antiphage defense systems. However, the mechanisms of action of most of these systems have exclusively been studied using phages with DNA genomes as the models, while phages with RNA genomes remain understudied. Here, we investigate how the defense system ApeA confers resistance against RNA phage infection. We show that two ApeA homologs, Ec1ApeA and Ps2ApeA, protect against a variety of single-stranded RNA phages. Focusing on Ec1ApeA, we find that it senses infection through a conserved pocket that likely binds an RNA structure in the phage genome. This activates the HEPN (higher eukaryotes and prokaryotes nucleotide-binding) RNase domain of Ec1ApeA which consequently cleaves the phage genomic RNA to restrict replication. In contrast to many other described defense systems, Ec1ApeA activity directly stops viral replication without inducing cell death, establishing ApeA as a non-abortive defense system that protects against RNA phages. Our results add to the increasingly diverse targets of antiviral HEPN RNases and provide insights into the understudied field of RNA phage defense.

Cardiomyocyte mitochondria align with sarcomeres during heart development. Mitochondrial motility is controlled by RHOT (ras homolog family member T) 1 and RHOT2. RHOT1 and RHOT2 are atypical Rho-like small GTPases that are anchored to the outer mitochondrial membrane and couple mitochondria to kinesin and dynein motors. We hypothesized that RHOT protein expression and mitochondrial motility are required for mitochondrial positioning during cardiomyocyte development. We generated mice with cardiomyocyte-selective deletion of Rhot1 and Rhot2 during embryogenesis (cRhot1/2-KO [constitutive and embryonic cardiomyocyte-selective Rhot1/2 knockout]) or tamoxifen-inducible deletion in the adult heart (iRhot1/2-KO [inducible cardiomyocyte-selective Rhot1/2 knockout]) to assess the importance of mitochondrial motility during and after cardiomyocyte maturation. Mitochondrial motility was determined by a motor protein-driven single mitochondria motility assay. Respiratory capacity was measured in isolated mitochondria. Intracellular mitochondrial localization and ATP production in isolated cardiomyocytes were assessed by confocal microscopy and after adenoviral expression of the fluorescence resonance energy transfer-based ATP biosensor ATeam. Cardiac ultrastructure was assessed by electron micrographs; mass spectrometry was used for proteome analysis. cRhot1/2-KO mice developed fatal cardiomyopathy associated with sarcomere disarray and perinuclear accumulation of mitochondria and ATP production. Mitochondria isolated from cRhot1/2-KO hearts exhibited impaired motility but preserved respiratory capacity. Mechanistically, proteome analysis identified that RHOT proteins bind mitochondria to contractile muscle fiber proteins. In contrast, inducible deletion of Rhot1 and Rhot2 in adult iRhot1/2-KO mice did not result in heart failure. Despite impaired motility of isolated mitochondria, intracellular mitochondrial localization, local ATP production, and sarcomere structure were preserved in adult iRhot1/2-KO hearts after cardiomyocyte maturation. RHOT proteins bind mitochondria to contractile muscle fiber proteins and are required for mitochondrial positioning in cardiomyocytes during development. Our study links mitochondrial motility and local ATP production to structural and functional maturation of the heart.

Talaromyces (Eurotiales, Trichocomaceae) is a monophyletic genus with species that impact the environment, human health, and various industries. Talaromyces consists of eight sections, and their ecology, diversity, and number of recognised species are rapidly expanding due to advances in morpho-molecular studies. In this study, we analyse the fungal diversity of soil samples collected from the Doi Tung Forest areas, which are dominated by Pinus kesiya in Chiang Rai, Thailand. Morphological and multi-gene phylogenetic analyses of internal transcribed spacer (ITS1-5.8S-ITS2), ß-tubulin (BenA), calmodulin (CaM), and the second largest subunit of the DNA-directed RNA polymerase II (rpb2) sequences were used to confirm the taxonomic placements of the new isolates. Herein, we describe Talaromyces doitungensis as a new species, report T. rubidus for the first time in Thailand (marking its second global record). Further, multi-gene phylogenetic analyses confirmed the distinct placement of these species and determined their relationships, supporting their recognition based on the phylogenetic species concept. In addition, preliminary antibacterial screening revealed that Talaromyces rubidus exhibited activity against Bacillus subtilis. Our findings contribute to the understanding of Talaromyces diversity and ecology by documenting a new species and new geographic records from pine-dominated forest soils in northern Thailand, and further studies are necessary to explore the bioactive compounds produced by Talaromyces species.

To understand how lung pathology relates to symptoms, microbiology, and progression risk in tuberculosis and to advance diagnostic development, we determined the frequency at screening of tuberculosis-consistent lesions in asymptomatic individuals within 5 years of tuberculosis diagnosis using highly sensitive imaging ([18F]-fluorodeoxyglucose PET-CT) and compared with chest x-ray computer-aided detection (CAD). We enrolled a prospective longitudinal cohort in Khayelitsha, Cape Town, South Africa, of asymptomatic, HIV-uninfected contacts aged 18-65 years of patients with rifampicin-resistant tuberculosis, a tuberculosis high-risk group not eligible for chemoprophylaxis. Participants underwent baseline PET-CT, chest x-ray, phlebotomy, and intensive sputum collection, and were classified into four PET-CT lung categories: consistent with tuberculosis, inactive tuberculosis, other lesions, and normal. Chest x-ray was processed by three types of CAD software (CAD4TB [version 7.0], qXR [version 3.0.0], and Lunit [version 3.1.4.111]). Follow-up included symptom-agnostic tuberculosis screening (23-38 months) and provincial register review (≤74 months), and a subgroup had repeat PET-CT (5-15 months). Tuberculosis was defined as bacteriologically confirmed or clinically diagnosed. The primary outcome measures were hazard ratio (HR) for tuberculosis diagnosis and treatment by baseline PET-CT lung abnormality category with normal as the reference group, and diagnostic performance of chest x-ray CAD software using area under the receiver operator characteristic curve (AUC). 250 asymptomatic adults were enrolled between March 3, 2015, and Oct 11, 2017, irrespective of tuberculosis history or previous infection, and followed up for 1107 person-years (median 4·7 years [IQR 4·0-5·1]). 18 (7%) participants were treated for tuberculosis (16 [89%] of 18 bacteriologically confirmed). Six of 18 participants were diagnosed at baseline (four requiring induced sputum culture) and 12 of 18 after a median of 32 months (IQR 12-35). By baseline PET-CT category, tuberculosis was diagnosed and treated in 12 (41%) of 29 participants with scans consistent with tuberculosis, two (7%) of 30 with scans consistent with inactive tuberculosis, two (2%) of 83 with scans showing other lesions, and two (2%) of 108 with scans showing normal lungs. Participants with baseline PET-CT scans consistent with tuberculosis had the highest risk of 5-year tuberculosis diagnosis (HR 28·54 [95% CI 6·37-127·81] compared with those with scans showing normal lungs, p<0·0001), with no significant risk for scans consistent with inactive tuberculosis (3·55 [0·50-25·21], p=0·21) or other lung lesions (1·30 [0·18-9·23], p=0·79). 11 (69%) of the 16 participants with bacteriologically confirmed tuberculosis were asymptomatic at bacteriological confirmation, and ten (91%) of 11 had baseline PET-CT scans consistent with tuberculosis. Using baseline PET-CT classification as reference, the AUC for chest x-ray CAD software ranged from 0·86 (95% CI 0·72-0·99) to 0·89 (0·75-1·00) for bacteriologically confirmed tuberculosis. Most adult asymptomatic contacts diagnosed with tuberculosis over 5 years had baseline radiographically evident disease, not radiographically negative incipient tuberculosis. Although PET-CT is not feasible for routine screening, it provides a highly sensitive reference benchmark for diagnostic development, with chest x-ray CAD performing comparatively well. South Africa Medical Research Council, US National Institutes of Health, Gates Foundation, Wellcome, UK Research and Innovation Medical Research Council, and Walter and Eliza Hall Institute of Medical Research.

Sheeppox virus (SPPV) is a major livestock pathogen causing economic hardship through reduced production and death of vulnerable sheep, with written descriptions of sheeppox-like disease recorded since antiquity. We report 21 novel ancient SPPV genomes spanning the Eurasian steppe Bronze Age (~1,700 BCE) to the Early Modern period in Western Europe, including multiple genomes obtained from medieval parchment. We estimate that major capripoxvirus lineages diverged ~11,500-3,700 years ago, overlapping known translocations and bio-cultural developments in sheep. Our dataset supports SPPV diverging first within the lineage leading to goatpox virus and lumpy skin disease virus, and that known gene inactivation events within SPPV and goatpox virus occur in our earliest SPPV genomes. These findings reveal that the food security of Eurasian communities have been threatened by sheeppox for over 3,700 years, and provide new insights to the genomic evolution and potential host adaptation of sheeppox virus.

The relationship between childhood maltreatment (CM) and obesity is nuanced, and recent evidence suggests stronger associations between CM and obesity-related traits in females compared to males. This study aims to validate and extend these findings in a large sample from the German National Cohort (NAKO). The NAKO is a population-based cohort study including 204,744 adults. For the present analyses, 151,143 individuals (74,596 female) were included. CM was assessed using the Childhood Trauma Screener (CTS). From the CTS, an overall severity score (CTS sum score), a cumulative CM score (number of CM subtypes with at least moderate severity), and five CTS subtypes were considered as exposures. Obesity-related traits included anthropometric (height, weight, body mass index [BMI], waist circumference [WC]) and body fat markers (relative fat mass [rFM], subcutaneous [SAT], visceral adipose tissue [VAT]). Sex-stratified linear and logistic regression models were adjusted for age, education, and examination center to associate CTS-based scores with obesity-related traits. Associations of the CTS sum score with weight, BMI, WC, rFM, and SAT were stronger in females compared to males, while similar associations were observed for VAT. In both sexes, most obesity-related traits exhibited dose-response relationships with increasing numbers of CM subtypes. Compared to unexposed females, females with exposure to ≥3 CM subtypes had a higher risk for obesity (i.e., BMI ≥ 30 kg/m2; OR = 1.56; 95% CI: 1.43, 1.71) and high WC (i.e., WC ≥ 88 cm; OR = 1.39; 95% CI: 1.29, 1.50). In males, exposure to ≥3 CM subtypes was also associated with increased obesity risk (OR = 1.51; 95% CI: 1.32, 1.72) and high WC (i.e., WC ≥ 102 cm; OR = 1.31; 95% CI: 1.18, 1.44). Physical and emotional abuse exhibited the strongest average associations and were associated with the most outcomes. Associations of CM exposure with adult anthropometric and body fat markers are stronger in females compared to males.

Capsular polysaccharides (CPS) are key virulence determinants, constituting the protective capsule that surrounds bacterial pathogens. Here, we present the complete cryo-EM structure of Gram-negative bacterial CPS secretion machinery formed by the E. coli K12 Wza-Wzc complex. The structure reveals an elongated, continuous channel spanning the entire envelope that facilitates CPS secretion. Multiple structural snapshots of the ADP-bound Wza-Wzc complex capture intermediate conformations of the double membrane assembly, highlighting its remarkable intrinsic dynamics. In-depth analysis of the isolated Wza translocon and Wzc co-polymerase, reveals mechanistic details of both complex formation and CPS transport. We further uncover the jellyroll domain of Wzc as a CPS-binding module, likely guiding CPS repeat units into a proposed Wzc-Wzy polymerization platform. Collectively, this work provides structural and functional insights into CPS synthesis and transport, advancing our understanding of bacterial capsule formation and virulence mechanisms.

In course of investigating the endophytic and nematode-associated fungus Polydomus karssenii for the production of secondary metabolites, seven previously undescribed natural products were isolated from liquid and solid-state fermentations. 1D and 2D NMR spectroscopy, together with HR-ESI–MS data, enabled the elucidation of the planar structures of 3-decalinoyltetramic acids polydosetins A–E (1–5) and cyclodepsipeptides pullularins G and H (6 and 7). The relative configurations of the decalin moiety of 1–5 were determined based on ROESY correlations and 1H–1H coupling constants. The configuration of the side chains was established through a detailed J-resolved analysis (Murata’s method) in combination with chemical shift comparison to model compounds. Absolute stereochemistry of 1–5 was assigned based on ECD data, and confirmed by Mosher’s method utilizing 3. Finally, the absolute configuration of amino acid residues in 6 and 7 was determined through advanced Marfey’s method. Bioassays revealed that compounds 1, 3, 5, and 7 were active against Gram-positive bacteria, 3 and 5 exhibited antifungal activity, and 1 and 2 showed nematicidal effects. These results underscore the untapped chemical potential of P. karssenii and highlight the importance of exploring nematode-associated fungi as sources of new natural products with potential antimicrobial and nematicidal properties.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13659-025-00579-8.

Abstract Inspired by natural gene transfer systems, we developed ACTIMOT, a CRISPR-Cas9–based in vivo DNA mobilization and multiplication platform that transfers large biosynthetic gene clusters (BGCs) from bacterial chromosomes onto replicative plasmids within the same cell. This strategy amplifies and activates cryptic BGCs efficiently, greatly enhancing product yields and enabling the discovery of four new natural product families, thereby unlocking hidden microbial biosynthetic potential.