Martin Alexander Fischer

Vancomycin-resistant Enterococcus faecium (VR Efm ) is a major cause of healthcare-associated infections, yet outbreak analysis remains challenging, particularly for prevalent lineages. One of the most striking examples is the Sequence Type (ST) 117/Complex Type (CT) 71/ vanB lineage, whose rapid expansion across Germany coincided with a sharp rise in VRE prevalence observed around 2019. Defining reliable genomic thresholds remains particularly difficult, as current core-genome Multi-Locus Sequence Typing (cgMLST) cutoffs are based largely on expert consensus or limited case series. Here, we evaluate the discriminatory performance of cgMLST and split k-mer analysis (SKA) for resolving relatedness within ST117/CT71 and investigate whether low core-genome diversity represents a lineage-specific trait or a population-wide property of E. faecium . While cgMLST captures the overall population structure of ST117/CT71, SKA provided higher resolution, distinguishing isolates identical by cgMLST. Analysis of 2,505 genomes showed that ST117/CT71 does not exhibit reduced allelic variability compared with other hospital-adapted lineages, suggesting that limited core-genome diversity is a broader population characteristic. Complementary transposon analyses further identified two temporally distinct ST117/CT71 sublineages carrying different Tn1549-like structures. To assess allelic variation within confirmed transmission events, we analyzed 138 outbreak clusters from 96 hospitals. Outbreak-associated isolates showed sharply compressed allelic differences (median = 1), with 90% of pairwise comparisons falling within ≤9 alleles. Thus, the commonly applied ≤20-allele threshold risks overestimating genomic relatedness and grouping unrelated isolates. Based on these data, we propose lowering the cgMLST alert threshold to ≤9 alleles for initial cluster detection of local outbreaks, followed by confirmatory high-resolution analyses such as SKA.

A significant increase of vancomycin‐resistant Enterococcus faecium (VREfm) infections was observed in South‐Eastern Austria since 2024. The prolonged outbreak is caused by a novel vanB‐ VREfm clone (ST117/CT7799, “VREfm _styr ”). This study characterizes the atypical difficult‐to‐detect resistance phenotype and assesses the genomic relatedness of the isolates. Patient and outbreak characteristics were investigated including whole genome sequencing of the isolates. Sensitivity of broth microdilution (BMD), gradient tests (GT), disk diffusion (DD), and automated susceptibility testing (VITEK2) was compared. The performance of commercial screening media was evaluated. From sporadic detections in early 2024 case numbers began to rise during the year. In 30/31 (97%) of all cases, intra‐hospital transmission was considered likely and an association with invasive procedures was identified in most cases. Core genome multilocus sequence typing revealed only six allelic differences between VREfm _styr isolates collected in a 12‐month period, all belonging to the E. faecium ST117/CT7799 lineage. BMD detected vancomycin resistance (MIC > 4 mg/L) in no more than 16/31 (52%) of isolates after 24 h incubation, while GT and DD misclassified all isolates. Only prolonged incubation improved the performance of these assays. VITEK2 analysis, however, correctly classified all 31 isolates. Of four commercially available VRE‐screening agars, only one was capable of detecting VREfm _styr after 24 h incubation. The emergence and clonal dissemination of VREfm ST117/CT7799 reveals a serious diagnostic gap as commonly used diagnostic algorithms fail to reliably detect this resistance phenotype. Our findings should help to further evaluate the true geographical distribution and clinical significance of this novel VREfm clone.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) cause infections, particularly nosocomially, with limited treatment options. NDM-1-producing Klebsiella pneumoniae cases have substantially increased since 2022, associated with the Ukraine war. AIM: We aimed to investigate transmission patterns using Germany’s Integrated Genomic Surveillance (IGS), combining notifications and sequence data. METHODS: We selected NDM-1-producing K. pneumoniae cases, confirmed by isolates between 1 January 2022 and 28 February 2023. Isolates were Illumina whole genome-sequenced and linked to notifications. Clusters were defined as ≤ 12 allelic differences in core genome-wide single nucleotide variant-based genotyping. Cluster categories were: ‘no exposure abroad’, ‘exposure in Ukraine’ or ‘other exposure abroad’ if ≥ one case stayed in Ukraine or elsewhere. Follow-up of 13 clusters examined further exposure information. RESULTS: Among 424 cases of most frequent sequence types, 326 (77%) belonged to 61 clusters. Seventeen (28%) clusters were associated with no exposure abroad, 33 (54%) with exposure in Ukraine, seven (11%) with other exposure abroad, and four (7%) had insufficient data. Cases in clusters with exposure in Ukraine were more dispersed, younger, and more often wound-infected than in other exposure location categories (p < 0.01). Cluster follow-up revealed one cluster with all cases from Ukraine or Russia, another with nosocomial transmission following case importation, and a third with all cases from one German hospital without exposure abroad. CONCLUSION: Most cases were in clusters, suggesting preventable chains of transmission. Three patterns emerged: transmission abroad, transmission in German hospitals from imported cases or local outbreaks. IGS can identify where transmission could be interrupted. International cooperation needs strengthening to prevent CPE spread.

Introduction: Vancomycin-resistant Enterococcus faecium (VREfm) isolates of sequence type (ST)1299 were described recently in south-eastern German hospitals and rapidly expanded from local to cross-border level. Aim: We describe the spread of the novel VREfm strain ST1299/vanA on a genetic, geographical and temporal level during the first 5 years after its detection. Methods: At University Hospital Regensburg (UHoR), routine VREfm surveillance is whole genome sequencing-based (≥ 1 VREfm per van-genotype, patient and year). In this observational cohort study, we analysed one VREfm ST1299 isolate from our database (2016–2022) per patient and year. Isolates were added from the Hospital of the Merciful Brothers Regensburg (MBR), the National Reference Centre for Staphylococci and Enterococci (NRC), and clinical isolates from Austria. Results: We identified 635 VREfm ST1299 isolates (100% vanA), including 504 from Regensburg, and 113 blood cultures. ST1299 isolates were first detected in 2018 simultaneously in Regensburg (n = 2) and southern Bavaria (n = 2), with local (UHoR) and regional numbers increasing rapidly from 2020, shifting to national scale in the same year. Genome data, analysed by cgMLST, showed a predominance of ST1299/CT1903 (315/504 isolates, 62.5%) and ST1299/CT3109 (127/504 isolates, 25.2%) isolates from Regensburg. By 2021, ST1299/CT1903 reached Upper Austria causing hospital outbreaks (n = 5). Phylogeny analysis suggests common ancestors with VREfm ST80, ST18 and ST17. Conclusion: Since their emergence in 2018, two highly transmissible subtypes of ST1299/vanA reached national, then cross-border scale. The observed outbreak tendency may explain the rapid and successful spread and the high clonality in our collection.

Metals are essential for all living organisms, but the type of metal and its concentration determines its action. Even low concentrations of metals may have toxic effects on organisms and therefore exhibit antimicrobial activities. In this study, we investigate the evolutionary adaptation processes of Staphylococcus aureus to metals and common genes for metal tolerance. Laboratory and clinical isolates were treated with manganese, cobalt, zinc, or nickel metal salts to generate growth-adapted mutants. After growth in medium supplemented with zinc, whole-genome sequencing identified, among others, two genes, mgtE (SAUSA300_0910) , a putative magnesium transporter and spoVG (SAUSA300_0475), a global transcriptional regulator, as hot spots for stress-induced single-nucleotide polymorphisms (SNPs). SNPs in mgtE were also detected in mutants treated with high levels of cobalt or nickel salts. To investigate the effect of these genes on metal tolerance, deletion mutants and complementation strains in an S. aureus USA300 LAC* laboratory strain were generated. Both, the mgtE and spoVG deletion strains were more tolerant to cobalt, manganese, and zinc. The mgtE mutant was also more tolerant to nickel exposure. Inductively coupled plasma mass spectrometry analysis demonstrated that the mgtE deletion mutant accumulated less intracellular zinc than the wild type, explaining increased tolerance. From these results, we conclude that mgtE gene inactivation increases zinc tolerance presumably due to reduced uptake of zinc. For the SpoVG mutant, no direct effect on the intracellular zinc concentration was detected, indicating toward different pathways to increase tolerance. Importantly, inactivation of these genes offers a growth advantage in environments containing certain metals, pointing toward a common tolerance mechanism. IMPORTANCE: Staphylococcus aureus is an opportunistic pathogen causing tremendous public health burden and high mortality in invasive infections. Treatment is becoming increasingly difficult due to antimicrobial resistances. The use of metals in animal husbandry and aquaculture to reduce bacterial growth and subsequent acquisition of metal resistances has been shown to co-select for antimicrobial resistance. Therefore, understanding adaptive mechanisms that help S. aureus to survive metal exposure is essential. Using a screening approach, we were able to identify two genes encoding the transporter MgtE and the transcriptional regulator SpoVG, which conferred increased tolerance to specific metals such as zinc when inactivated. Further testing showed that the deletion of mgtE leads to reduced intracellular zinc levels, suggesting a role in zinc uptake. The accumulation of mutations in these genes when exposed to other metals suggests that inactivation of these genes could be a common mechanism for intrinsic tolerance to certain metals.

Background: The war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients. Aim: To investigate the genomic epidemiology of New Delhi metallo-β-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries. Methods: Whole-genome sequencing of 66 isolates sampled in 2022–2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla _NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla _NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire. Results: wgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla _NDM-1-carrying-P. stuartii and the third bla _NDM-5-carrying-P. stuartii. The bla _NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla _NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla _NDM-1, bla _OXA-10, bla _CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam–avibactam. Patients with data on age (n = 41) were 19–74 years old; of 49 with information on sex, 38 were male. Conclusion: XDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

The National Reference Centre for Enterococci receives an increasing number of linezolid-resistant Enterococcus isolates. Linezolid (LIN) resistance is mediated by G2576T 23S rDNA gene mutations and/or acquisition of resistance genes (cfr, optrA, poxtA). There are anecdotal reports that those resistance traits may be present in phenotypically linezolid-susceptible isolates. We aimed to determine the prevalence of LIN resistance genes and mutations in enterococci with a LIN MIC of 4 mg/L in broth microdilution (EUCAST = susceptible) isolated from German hospital patients 2019–2021. LIN MICs were additionally determined by ETEST^® and VITEK2. Selected strains were subjected to LIN selective pressure and growth was monitored with increasing antibiotic concentrations. We received 195 isolates (LIN MIC = 4 mg/L). In total, 78/195 (40%) isolates contained either a putative resistance gene, the G2576T mutation, or a combination thereof. Very major error was high for broth microdilution. The ability to predict phenotypic resistance from genotypic profile was highest for G2576T-mediated resistance. Selection experiments revealed that, in particular, E. faecium isolates with resistance gene mutations or poxtA rapidly adapt to MICs above the clinical breakpoint. In conclusion, LIN resistance genes and mutations can be observed in phenotypically linezolid-susceptible enterococci. Those isolates may rapidly develop resistance under LIN selective pressure potentially leading to treatment failure.

A Gram-positive, motile, aerobic, rod-shaped, endospore-forming strain designated IRB4-01T was isolated from fermented African locust beans (Iru) obtained from Bodija market in the city of Ibadan, southwestern Nigeria, during a screening process from food-related sources. IRB4-01T grew at 10–50 °C (optimum, 35–37 °C), pH 6–10 (optimum, pH 7) and in 0–6 % NaCl (optimum, 1–3 %). Phylogenetic analyses based on 16S rRNA and combined short- and long-read genome sequencing revealed that IRB4-01T is closely related to Lysinibacillus cavernae SYSU K30005T and Lysinibacillus boronitolerans 10aT. The cell-wall peptidoglycan type was A4α (Lys–Asp), containing the diagnostic diamino acid lysine. The major polar lipids in strain IRB4-01T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid, while the predominant menaquinone was MK-7. The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. Genomic DNA G+C content was 37.4 mol%, while the digital DNA–DNA hybridization revealed 33.6 and 32.3 % relatedness to L. cavernae SYSU K30005T and L. boronitolerans 10aT, respectively. Based on phenotypic, physiological and chemotaxonomic characteristics, as well as genome comparisons, strain IRB4-01T represents a novel species of the genus Lysinibacillus , for which the name Lysinibacillus irui sp. nov. is proposed. The type strain is IRB4-01T (NCIMB 15452T=LMG 32887T). Hybrid genome data are provided on the NCBI database using the Bioproject number PRJNA906010 and accession numbers CP113527 and CP113528. Additionally, a representative 16S rRNA sequence is available with the GenBank accession number OQ566940.

The acquisition of hypervirulence-associated genes by carbapenemase-producing Klebsiella pneumoniae is being increasingly observed, and easy-to-use diagnostic tests are needed for the surveillance of the hypervirulent K. pneumoniae (hvKp). In this pilot study, 87 K. pneumoniae isolates from invasive infections collected in 2022 and 2023 were analysed using the LAMP-based eazyplex^® Superbug CRE and hvKp assays for the simultaneous identification of carbapenemases and virulence genes (rmpA/A2, iuC, iroC, ybt, clb). Nine isolates showed a Kleborate virulence score of 4 or 5 (10.3%). The time for the results of the eazyplex^® assays ranged from 6.5 to 13 min, and the total turnaround time, including sample preparation, was less than 30 min. Five isolates, three of which produced New Delhi metallo-beta lactamase (NDM), were subjected to whole-genome sequencing (WGS) analysis for further characterisation. The eazyplex^® test results for beta-lactamase and virulence genes were confirmed. The eazyplex^® hvKp, currently only available as a Research Use Only assay, may be a useful tool for the rapid identification of hvKp without significant additional workload when combined with the eazyplex^® Superbug CRE assay for the detection of carbapenemases.

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.