Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

Background: Hepatitis C virus (HCV) constitutes a global health problem, while hepatitis E virus (HEV) is the major cause of acute viral hepatitis globally. HCV/HEV co-infections have been poorly characterized, as they are hampered by the lack of robust HEV cell culture systems. This study developed experimental models to study HCV/HEV co-infections and investigate viral interference in cells and humanized mice. Methods: We used state-of-the art human hepatocytes tissue culture models to assess HEV and HCV replication in co- or super-transfection settings. Findings were confirmed by co- and super-infection experiments in human hepatocytes and in vivo in human liver chimeric mice. Results: HEV was inhibited by concurrent HCV replication in human hepatocytes. This exclusion phenotype was linked to the protease activity of HCV. These findings were corroborated by the fact that in HEV on HCV super-infected mice, HEV viral loads were reduced in individual mice. Similarly, HCV on HEV super-infected mice showed reduced HCV viral loads. Conclusion: Direct interference of both viruses with HCV NS3/4A as the determinant was observed. In vivo, we detected reduced replication of both viruses after super-infection in individual mice. These findings provide new insights into the pathogenesis of HCV-HEV co-infections and should contribute to its clinical management in the future.

Vancomycin-resistant enterococci (VRE) occur in hospitalized patients, causing both infection and colonization. In recent years, there has been an increase in VRE in German and other hospitals, raising the question of how to control this epidemic best. To better understand the specific epidemiology and to guide infection control, we conducted a retrospective cohort study analyzing all patients with VRE at Hannover Medical School, a tertiary university clinic in Germany that specializes in solid organ transplantation. Epidemiologic and clinical characteristics of patients with VRE from 2015–2017 were collected. Basic epidemiologic parameters, including VRE incidence and incidence density, were calculated. Independent risk factors for nosocomial VRE infection compared to colonization were assessed using a logistic regression model. There were 1,492 VRE cases corresponding to 822 individual patients. The incidence was 0.8 VRE cases per 100 cases. A total of 536 (35.9%) of the 1,492 VRE cases were acquired nosocomially. Of the 1,492 cases, 912 cases had VRE-positive samples (894 Enterococcus (E.) faecium and 18 E. faecalis) in our hospital laboratory and the remaining cases were known VRE carriers. The vanB-phenotype was observed in 369 of the 894 (41.3%) E. faecium isolates and in 6 of the 18 (33.3%) E. faecalis isolates. There was an increase over time in the vanB-phenotype proportion in E. faecium (2015: 63 of 171, 36.8%, 2016: 115 of 322, 35.7% and 2017: 191 of 401, 47.6%). A total of 107 cases had a VRE infection (7.2% of all VRE cases) according to the criteria of the German National Reference Center for Surveillance of Nosocomial Infections. The remaining cases were only colonized. Among other factors, leukocytopenia (<1,000/μL), the use of a central venous catheter and the visceral surgery medical specialty were independently associated with nosocomial VRE infection. VRE imposed a relevant and increasing infection control burden at our hospital. Nosocomial VRE infection was predominantly found in certain medical specialties, such as hematology and oncology and visceral surgery. Infection control efforts should focus on these highly affected patient groups/specialties.

In preterm and term infants who require intermediate or intensive care Methicillin-resistant Staphylococcus aureus (MRSA) infection can lead to significant morbidity. In this study MRSA colonization and infection were assessed in a mixed tertiary neonatal intensive and intermediate care unit in Germany over an 8-year period (2013–2020). We investigated patient-related factors, associated with nosocomial MRSA acquisition, and we discuss our infection control concept for MRSA. Of 3488 patients treated during the study period, 24 were MRSA positive patients, corresponding to 26 patient hospital stays. The incidence was 0.7 MRSA patients per 100 patients. The incidence density was 0.4 MRSA patient hospital stays per 1000 patient days. Twelve patients (50%) acquired MRSA in the hospital. One patient developed a hospital acquired MRSA bloodstream infection 9 days after birth (i.e., 0.03% of all patients on the ward during the study period). A total of 122 patients had to be screened to detect one MRSA positive patient. In a logistic regression model, the use of 3^rd generation intravenous cephalosporin (cefotaxim) was associated with nosocomial MRSA acquisition compared with matched control patients who did not acquire MRSA. In sum, the burden of MRSA colonization and infection in the ward was low during the study period. A comprehensive infection control concept that included microbiologic colonization screening, prospective infection surveillance together with isolation and emphasis on basic hygiene measures is essential to handle MRSA in this specialized setting.

A substantial and rapid increase, followed by a sharp decline in vanB -type vancomycin-resistant Enterococcus faecium (VRE), occurred in Germany in the late 2010s. This unusual epidemiological trend prompted detailed genomic investigations to explore the underlying dynamics at a German university hospital. We retrospectively analyzed 344 E. faecium bloodstream infection (BSI) isolates collected between 2017 and 2022. Isolates were classified as vanA -positive, vanB -positive, or van- negative, while VRE was defined as vanA - or vanB -positive. Molecular typing included multilocus sequence typing (MLST), core genome MLST (cgMLST), and split k-mer analysis (SKA). Clonal relationships and potential patient-to-patient transmission events were assessed by integrating genomic data with machine-aided analysis of available longitudinal patient movement data. High-resolution genomic analysis of BSI isolates revealed an oligoclonal scenario involving multiple epidemic lineages (e.g., sequence type (ST)117/complex type (CT)71, ST117/CT929, ST117/CT2505, ST80, and ST262) with distinct van genotypes and dynamic changes over time. Overall, genomic overlap between vanA -positive, vanB -positive, and van -negative populations was minimal. The vanB surge, peaking in late 2018 to early 2019, was mainly driven by ST117/CT71, ST117/CT36, and ST117/CT1917. SKA provided enhanced discriminatory power over cgMLST. The inferred transmission events linked bloodstream infections separated by long intervals (median, 106 days). Accurate characterization of the transient vanB -type VRE peak required an integrated genomic approach combining cgMLST, high-resolution SKA, and epidemiological data. These comprehensive methods enable infection prevention and control teams to distinguish true outbreaks from apparent epidemiologic clusters of polyclonal isolates. This allows for accurate interpretation and precisely targeted interventions where evidence supports transmission. IMPORTANCE: Vancomycin-resistant Enterococcus faecium is an increasingly important nosocomial pathogen worldwide. Understanding its epidemiology and transmission dynamics is critical to effectively control its spread. This study documents shifting E. faecium populations within a high-risk hospital environment, including both vancomycin-resistant and vancomycin-susceptible bloodstream isolates. Virulence gene profiling demonstrated that hospital-associated variants predominated across all major lineages, independent of vancomycin resistance, indicating that hospital adaptation is a common feature of both resistant and susceptible populations. Detailed genomic analyses, combining core genome multilocus sequence typing with high-resolution split k-mer analysis, integrated with comprehensive epidemiological tracking, were critical to accurately depict complex epidemiological dynamics. This combined approach allows precise differentiation between monoclonal outbreaks and oligoclonal transmission, enabling more targeted infection prevention and control strategies.

Background: Klebsiella pneumoniae complex (Kp) is a relevant neonatal pathogen colonizing preterm infants. While outbreak investigations often focus on multidrug-resistant strains, the epidemiology and genomic dynamics of wild-type Kp in nonoutbreak neonatal intensive care unit (NICU) settings remain elusive. Methods: We conducted a 30-month (October 2021 to March 2024) cohort study with weekly active, unselective colonization surveillance of all NICU patients to identify risk factors for nosocomial Kp acquisition and drivers of transmission in a tertiary 21-bed NICU/intermediate care unit (IMC) in Germany. Results: Among 936 patients, 8.7% carried Kp, of which 70.4% were nosocomial. Very low birth weight (VLBW; < 1500 g) was the only independent risk factor for nosocomial acquisition (adjusted odds ratio [aOR], 3.42; 95% CI, 1.29–9.32). Kp infections occurred in three Kp carriers (3.7%). Genomic analyses of at least the first isolate per patient (83 in total) revealed an oligoclonal population structure, with ten distinct sequence types (STs) underlying temporally overlapping clusters. Ten genomic clusters (median size, four patients) were identified, with markedly higher odds in VLBW infants (aOR, 8.76; 95% CI, 2.45–34.16). Nosocomial cluster-assigned cases had higher rates and longer durations of noninvasive ventilation and peripheral venous catheter use. Cluster prevalence showed climate-associated variation, with a six-feature extreme gradient boosting (XGBoost) model identifying temperature and humidity among the strongest predictors. Conclusions: Patient- and climate-associated parameters are main drivers of nosocomial wild-type Kp acquisition and cluster occurrence. Comprehensive surveillance and risk-adapted infection prevention and control support sustainable Kp control in VLBW infants.