Establishment of a Novel Short Tandem Repeat Typing Method for Exophiala dermatitidis.

The opportunistic black yeast-like fungus Exophiala dermatitidis frequently colonizes the respiratory tract of cystic fibroses (CF) patients. Additionally, it can cause superficial, systemic, and cerebral forms of phaeohyphomycoses. The objective of this study was to develop and apply a microsatellite or short tandem repeat (STR) genotyping scheme for E. dermatitidis. In total, 82 E. dermatitidis isolates from various geographic origins (environmental = 9, CF = 63, invasive isolates = 9, melanin-deficient mutant = 1) were included in this study. After next-generation sequencing of a reference strain and sequence filtering for microsatellites, six STR markers were selected and amplified in two multiplex PCR reactions. The included isolates were discriminated in a genetic cluster analysis using the Pearson algorithm to reveal the relatedness of the isolates. The E. dermatitidis isolates clustered on basis of both, their source and their origin. The invasive isolates from Asia were unrelated to isolates from CF. Nearly all environmental isolates were grouped separately from patients' isolates. The Simpson index was 0.94. In conclusion, we were able to establish a STR genotyping scheme for investigating population genomics of E. dermatitidis.

Targeted PCR of Mucorales in pediatric bronchoalveolar lavage samples indicates low prevalence of airway colonization and sample contamination.

Mucorales are a large order of ubiquitous saprophytic zygomycete fungi and act as opportunistic pathogens in humans. In pediatric patients, little is known about the role of Mucorales in airway colonization and infection or their role as contaminants of respiratory samples. Currently, polymerase chain reaction (PCR) is the most sensitive mode of detection Mucorales in clinical specimen. In this study, we aimed to determine the prevalence of Mucorales in bronchoalveolar lavage samples (BAL) from a large, diverse group of pediatric patients. We performed commercial Mucorales PCR (MucorGenius®, Pathonostics, Maastricht, NL, USA) on 102 thawed BAL samples of 100 patients. Mucorales PCR was negative in all samples. Our data suggest that Mucorales spp. have a low prevalence in paediatric airways and do not frequently contaminate pediatric BAL samples.

Diagnostic Accuracy of Multiplex Polymerase Chain Reaction in Early Onset Neonatal Sepsis.

Early onset neonatal sepsis is a significant contributor to neonatal morbidity and mortality. Although blood cultures remain the diagnostic gold standard, they detect pathogens in only a minority of suspected cases. This study compared the accuracy of blood cultures with a rapid multiplex PCR test. Newborns at risk of neonatal sepsis were prospectively screened as recommended per national guidelines. Evaluations included laboratory parameters (CrP, IL6, differential blood count), blood culture, and a molecular multiplex PCR test (ROCHE LightCycler SeptiFast®) identifying 20 common microbial agents. Blood samples were taken simultaneously from umbilical cord or venous sources on the first day of life. Of 229 infants included, 69% were born preterm. Blood culture and multiplex PCR sensitivity were 7.4% and 14.8%, respectively. Specificity, negative and positive predictive values between methods showed no significant variance, although multiplex PCR had more false positives due to contamination. The limited sensitivity of blood cultures for early onset neonatal sepsis is concerning. Despite quicker results, multiplex PCR does not enhance diagnostic accuracy or antibiotic therapy guidance, thus it cannot be recommended for this indication.

Microbiological Non-Culture-Based Methods for Diagnosing Invasive Pulmonary Aspergillosis in ICU Patients.

The diagnosis of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is crucial since most clinical signs are not specific to invasive fungal infections. To detect an IPA, different criteria should be considered. Next to host factors and radiological signs, microbiological criteria should be fulfilled. For microbiological diagnostics, different methods are available. Next to the conventional culture-based approaches like staining and culture, non-culture-based methods can increase sensitivity and improve time-to-result. Besides fungal biomarkers, like galactomannan and (1→3)-ß-D-glucan as nonspecific tools, molecular-based methods can also offer detection of resistance determinants. The detection of novel biomarkers or targets is promising. In this review, we evaluate and discuss the value of non-culture-based microbiological methods (galactomannan, (1→3)-ß-D-glucan, Aspergillus PCR, new biomarker/targets) for diagnosing IPA in ICU patients.

Targeting the Granulocytic Defense against A. fumigatus in Healthy Volunteers and Septic Patients.

Neutrophil granulocytes (NGs) are among the key players in the defense against Aspergillus fumigatus (A. fumigatus). To better elucidate a pathophysiological understanding of their role and functions, we applied a human cell model using NGs from healthy participants and septic patients to evaluate their inhibitory effects on the growth of A. fumigatus ex vivo. Conidia of A. fumigatus (ATCC® 204305) were co-incubated with NGs from healthy volunteers or septic patients for 16 h. A. fumigatus growth was measured by XTT assays with a plate reader. The inhibitory effect of NGs on 18 healthy volunteers revealed great heterogeneity. Additionally, growth inhibition was significantly stronger in the afternoon than the morning, due to potentially different cortisol levels. It is particularly interesting that the inhibitory effect of NGs was reduced in patients with sepsis compared to healthy controls. In addition, the magnitude of the NG-driven defense against A. fumigatus was highly variable among healthy volunteers. Moreover, daytime and corresponding cortisol levels also seem to have a strong influence. Most interestingly, preliminary experiments with NGs from septic patients point to a strongly diminished granulocytic defense against Aspergillus spp.

Diagnosis and Treatment of Invasive Aspergillosis Caused by Non-fumigatus Aspergillus spp.

With increasing frequency, clinical and laboratory-based mycologists are consulted on invasive fungal diseases caused by rare fungal species. This review aims to give an overview of the management of invasive aspergillosis (IA) caused by non-fumigatus Aspergillus spp.-namely A. flavus, A. terreus, A. niger and A. nidulans-including diagnostic and therapeutic differences and similarities to A. fumigatus. A. flavus is the second most common Aspergillus spp. isolated in patients with IA and the predominant species in subtropical regions. Treatment is complicated by its intrinsic resistance against amphotericin B (AmB) and high minimum inhibitory concentrations (MIC) for voriconazole. A. nidulans has been frequently isolated in patients with long-term immunosuppression, mostly in patients with primary immunodeficiencies such as chronic granulomatous disease. It has been reported to disseminate more often than other Aspergillus spp. Innate resistance against AmB has been suggested but not yet proven, while MICs seem to be elevated. A. niger is more frequently reported in less severe infections such as otomycosis. Triazoles exhibit varying MICs and are therefore not strictly recommended as first-line treatment for IA caused by A. niger, while patient outcome seems to be more favorable when compared to IA due to other Aspergillus species. A. terreus-related infections have been reported increasingly as the cause of acute and chronic aspergillosis. A recent prospective international multicenter surveillance study showed Spain, Austria, and Israel to be the countries with the highest density of A. terreus species complex isolates collected. This species complex seems to cause dissemination more often and is intrinsically resistant to AmB. Non-fumigatus aspergillosis is difficult to manage due to complex patient histories, varying infection sites and potential intrinsic resistances to antifungals. Future investigational efforts should aim at amplifying the knowledge on specific diagnostic measures and their on-site availability, as well as defining optimal treatment strategies and outcomes of non-fumigatus aspergillosis.

In Vitro and In Vivo Effect of the Imidazole Luliconazole against Lomentospora prolificans and Scedosporium spp.

Infections with Scedosporium spp. and Lomentospora prolificans have become a serious threat in clinical settings. The high mortality rates associated with these infections can be correlated with their multidrug resistance. The development of alternative treatment strategies has become crucial. Here, we investigate the in vitro and in vivo activity of luliconazole (LLCZ) against Scedosporium apiospermum (including its teleomorph Pseudallescheria boydii) and Lomentospora prolificans. The LLCZ MICs were determined for a total of 37 isolates (31 L. prolificans isolates, 6 Scedosporium apiospermum/P. boydii strains) according to EUCAST. Furthermore, the LLCZ antifungal activity was tested in vitro, using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] growth kinetics assay and biofilm assays (crystal violet and XTT assay). In addition, a Galleria mellonella infection model was used for in vivo treatment assays. The MIC90 of LLCZ was determined to be 0.25 mg/L for all tested pathogens. Growth was inhibited within 6 to 48 h of the start of incubation. LLCZ inhibited biofilm formation in both preadhesion stages and late-stage adhesion. In vivo, a single dose of LLCZ increased the survival rate of the larvae by 40% and 20% for L. prolificans and Scedosporium spp., respectively. This is the first study demonstrating LLCZ activity against Lomentospora prolificans in vitro and in vivo and the first study showing the antibiofilm effect of LLCZ in Scedosporium spp. IMPORTANCE Lomentospora prolificans and S. apiospermum/P. boydii are opportunistic, multidrug-resistant pathogens causing invasive infections in immunosuppressed patients and sometimes in healthy persons. Lomentospora prolificans is panresistant against the currently available antifungals, and both species are associated with high mortality rates. Thus, the discovery of novel antifungal drugs exhibiting an effect against these resistant fungi is crucial. Our study shows the effect of luliconazole (LLCZ) against L. prolificans and Scedosporium spp. in vitro, as well as in an in vivo infection model. These data reveal the previously unknown inhibitory effect of LLCZ against L. prolificans and its antibiofilm effect in Scedosporium spp. It represents an extension of the literature regarding azole-resistant fungi and could potentially lead to the development of future treatment strategies against these opportunistic fungal pathogens.

Next Generation Sequencing of Free Microbial DNA for Rapid Identification of Pathogens in Critically Ill Children with Systemic Inflammatory Response Syndrome (SIRS).

BACKGROUND: Infections, major surgeries, and hyperinflammatory syndromes are known to trigger Systemic Inflammatory Response Syndrome (SIRS). Discrimination between infectious and noninfectious inflammation often poses a challenge in chronically ill patients with multiple comorbidities. These patients are routinely treated with a variety of anti-infective medications before a pathogen is identified. With the goal of improving pathogen detection rates and interventions, we evaluated Next Generation Sequencing (NGS) as a highly sensitive and fast means of detecting free microbial DNA in a small amount of serum samples from children with ongoing SIRS. METHODS: We describe seven complex pediatric patients of SIRS or prolonged fever (>38.5 °C) >72 hours in which serum samples analyzed by NGS had a major impact on therapy. One patient was analyzed twice. RESULTS: In eight NGS there were six positive results (two bacterial, three viral, one fungal) which were subsequently confirmed by microbiological culture or polymerase chain reaction (PCR) in five of the six NGS. In five of the eight performed NGS, results led to a change of therapy: antibiotic therapy was discontinued in two, escalated in one, an initiated in another; in one an antiviral was administered. CONCLUSIONS: NGS may become a valuable addition to infectious disease diagnostics in cases of pediatric SIRS. However, NGS has not yet been validated as a diagnostic method in pediatric as a diagnostic method in pediatric patients and results should therefore be interpreted with caution. Multi-center NGS evaluation studies are currently being planned.

Effect of Dexamethasone on the Incidence and Outcome of COVID-19 Associated Pulmonary Aspergillosis (CAPA) in Critically Ill Patients during First- and Second Pandemic Wave-A Single Center Experience.

Superinfections with Aspergillus spp. in patients with Coronavirus disease 2019 (CAPA: COVID-19-associated pulmonary aspergillosis) are increasing. Dexamethasone has shown beneficial effects in critically ill COVID-19 patients. Whether dexamethasone increases the risk of CAPA has not been studied exclusively. Moreover, this retrospective study aimed to identify risk factors for a worse outcome in critically ill COVID-19 patients. Data from 231 critically ill COVID-19 patients with or without dexamethasone treatment from March 2020 and March 2021 were retrospectively analysed. Only 4/169 (6.5%) in the DEXA-group and 13/62 (7.7%) in the Non-DEXA group were diagnosed with probable CAPA (p = 0.749). Accordingly, dexamethasone was not identified as a risk factor for CAPA. Moreover, CAPA was not identified as an independent risk factor for death in multivariable analysis (p = 0.361). In contrast, elevated disease severity (as assessed by Sequential Organ Failure Assessment [SOFA]-score) and the need for organ support (kidney replacement therapy and extracorporeal membrane oxygenation [ECMO]) were significantly associated with a worse outcome. Therefore, COVID-19 treatment with dexamethasone did not increase the risk for CAPA. Moreover, adequately treated CAPA did not represent an independent risk factor for mortality. Accordingly, CAPA might reflect patients' severe disease state instead of directly influencing outcome.

Sphingosine as a New Antifungal Agent against Candida and Aspergillus spp.

This study investigated whether sphingosine is effective as prophylaxis against Aspergillus spp. and Candida spp. In vitro experiments showed that sphingosine is very efficacious against A. fumigatus and Nakeomyces glabrataa (formerly named C. glabrata). A mouse model of invasive aspergillosis showed that sphingosine exerts a prophylactic effect and that sphingosine-treated animals exhibit a strong survival advantage after infection. Furthermore, mechanistic studies showed that treatment with sphingosine leads to the early depolarization of the mitochondrial membrane potential (Δψm) and the generation of mitochondrial reactive oxygen species and to a release of cytochrome C within minutes, thereby presumably initiating apoptosis. Because of its very good tolerability and ease of application, inhaled sphingosine should be further developed as a possible prophylactic agent against pulmonary aspergillosis among severely immunocompromised patients.

Multiple colony antifungal susceptibility testing detects polyresistance in clinical Candida cultures: a European Confederation of Medical Mycology excellence centers study.

OBJECTIVES: Many factors influence the outcome of in vitro antifungal susceptibility testing (AFST), including endpoint definition, inoculum sizes, time and temperature of incubation, and growth medium used. This European Confederation of Medical Mycology (ECMM) Excellence center driven study investigated multiple colony testing (MCT) of five separate colonies to investigate the prevalence of polyresistance (PR), defined as heterogeneous MICs from a same-species Candida culture irrespective of the underlying resistance mechanism. METHODS: Candida spp. MCT for fluconazole and anidulafungin was performed by Etest prospectively comprising 405 clinical samples. MCT results were compared to the real-life routine MIC data and PR was assessed. Candida colonies displaying strong PR were selected for genotyping using multilocus sequence typing and random amplified polymorphic DNA assays for C. lusitaniae. RESULTS: Candida PR was observed in 33 of 405 samples (8.1%), with higher rates for non-albicans species (26/186, 14%) than for C. albicans (7/219, 3.2%), and for fluconazole than for anidulafungin. MCT detected acquired resistance more often than routine AFST (18/405, 4.5%) and 9 of the 161 investigated blood cultures showed PR (5.6%). Multilocus sequence typing and random amplified polymorphic DNA did not reveal a uniform genetic correlate in strains studied. CONCLUSIONS: This study shows that Candida single MIC-values obtained in routine diagnostics may be incidental, as they fail to detect PR and resistant subpopulations reliably. The reasons for PR seem to be manifold and should be regarded as a phenotypical expression of genomic variability irrespective of the underlying resistance mechanism, which may help to interpret ambiguous and non-reproducible AFST results.

In Vitro and In Vivo Activity of Luliconazole (NND-502) against Planktonic Cells and Biofilms of Azole Resistant Aspergillus fumigatus.

Aspergillus fumigatus has become a significant threat in clinical settings. Cases of invasive infections with azole-resistant A. fumigatus isolates (ARAF) increased recently. Developing strategies for dealing with ARAF has become crucial. We here investigated the in-vitro and in-vivo activity of the imidazole luliconazole (LLCZ) against clinical ARAF. In total, the LLCZ minimum inhibitory concentrations (MICs) were tested for 101 A. fumigatus isolates (84 ARAF and 17 azole-susceptible A. fumigatus as wild-type controls) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Additionally, antifungal activity was assessed in vitro, including an XTT planktonic growth kinetics assay and biofilm assays (crystal violet and XTT assay). Further, a single-dose LLCZ treatment (152 mg/L) was tested for seven days in vivo in a Galleria mellonella infection model. LLCZ showed an MIC50 of 0.002 mg/L and no significant difference was found between triazole-resistant and wild-type isolates. Growth inhibition took place between 6 and 12 h after the start of incubation. LLCZ inhibited biofilm formation when added in the pre-adhesion stages. In vivo, single-dose LLCZ-treated larvae show a significantly higher survival percentage than the control group (20%). In conclusion, LLCZ has activity against planktonic cells and early biofilms of ARAF.

Prevalence of COVID-19 Associated Mucormycosis in a German Tertiary Care Hospital.

Due to Coronavirus disease (COVID-19) a new group of patients at risk emerged with COVID-19-associated mucormycosis (CAM). Systematic studies, evaluating the prevalence of CAM are missing. To assess CAM prevalence in a tertiary care hospital in Germany, we applied direct microscopy, fungal culture and quantitative realtime in-house PCR targeting Mucorales-specific fragments of 18S and 28S rRNA on respiratory specimens of 100 critically ill COVID-19 patients. Overall, one Mucorales-PCR positive bronchoalevolar lavage was found whereas direct microscopy and fungal culture were negative in all cases. We conclude that a routine screening for CAM in Germany is not indicated.

Inhibition of azole-resistant Aspergillus fumigatus biofilm at various formation stages by antifungal drugs, including olorofim.

OBJECTIVES: Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents. METHODS: Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48 h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed. RESULTS: Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12 h after incubation. CONCLUSIONS: This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus.

Antibacterial Activity of the Novel Drug Gepotidacin against Stenotrophomonas maltophilia-An In Vitro and In Vivo Study.

Stenotrophomonas maltophilia is increasingly recognized as a nosocomial bacterial pathogen with a multi-drug resistance profile. In this study, the novel drug gepotidacin, the first compound of the novel triazaacenaphthylene topoisomerase inhibitor antibiotics class, was evaluated on its activity against clinical S. maltophilia isolates. Ninety-nine S. maltophilia isolates plus reference strain K279a (N = 100) were tested on their susceptibility towards gepotidacin in a broth microdilution. Additional susceptibility testing was performed towards the commonly applied combination trimethoprim/sulfamethoxazole (TMP/SXT), moxifloxacin, and levofloxacin. The time-kill kinetic of gepotidacin was observed in a time-kill assay. The greater wax moth Galleria mellonella was used to determine the activity of gepotidacin against S. maltophilia in vivo. Gepotidacin showed minimum inhibitory concentrations (MICs) between 0.25 and 16 mg/L (MIC50: 2 mg/L; MIC90: 8 mg/L), independently of its susceptibility towards TMP/SXT. The five TMP/SXT resistant strains exhibited gepotidacin MICs from 1 to 4 mg/L. The S. maltophilia strains resistant to the assessed fluoroquinolones showed in parts high MICs of gepotidacin. The time-kill assay revealed a time- and strain-dependent killing effect of gepotidacin. In vivo, injection of gepotidacin increased the survival rate of the larvae from 61 % to 90 % after 2 days. This study showed antimicrobial effects of gepotidacin towards S. maltophilia.

COVID-19-associated pulmonary aspergillosis in ICU patients in a German reference centre: Phenotypic and molecular characterisation of Aspergillus fumigatus isolates.

BACKGROUND: COVID-19-associated invasive pulmonary aspergillosis (CAPA) is associated with increased mortality. Cases of CAPA caused by azole-resistant Aspergillus fumigatus strains have been reported. OBJECTIVES: To analyse the twelve-month CAPA prevalence in a German tertiary care hospital and to characterise clinical A. fumigatus isolates from two German hospitals by antifungal susceptibility testing and microsatellite genotyping. PATIENTS/METHODS: Retrospective observational study in critically ill adults from intensive care units with COVID-19 from 17 February 2020 until 16 February 2021 and collection of A. fumigatus isolates from two German centres. EUCAST broth microdilution for four azole compounds and microsatellite PCR with nine markers were performed for each collected isolate (N = 27) and additional for three non-COVID A. fumigatus isolates. RESULTS: welve-month CAPA prevalence was 7.2% (30/414), and the rate of azole-resistant A. fumigatus isolates from patients with CAPA was 3.7% with detection of one TR34/L98H mutation. The microsatellite analysis revealed no major clustering of the isolates. Sequential isolates mainly showed the same genotype over time. CONCLUSIONS: Our findings demonstrate similar CAPA prevalence to other reports and a low azole-resistance rate. Genotyping of A. fumigatus showed polyclonal distribution except for sequential isolates.

Global guideline for the diagnosis and management of the endemic mycoses: an initiative of the European Confederation of Medical Mycology in cooperation with the International Society for Human and Animal Mycology.

The global burden of the endemic mycoses (blastomycosis, coccidioidomycosis, emergomycosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and talaromycosis) continues to rise yearly and these infectious diseases remain a leading cause of patient morbidity and mortality worldwide. Management of the associated pathogens requires a thorough understanding of the epidemiology, risk factors, diagnostic methods and performance characteristics in different patient populations, and treatment options unique to each infection. Guidance on the management of these infections has the potential to improve prognosis. The recommendations outlined in this Review are part of the "One World, One Guideline" initiative of the European Confederation of Medical Mycology. Experts from 23 countries contributed to the development of these guidelines. The aim of this Review is to provide an up-to-date consensus and practical guidance in clinical decision making, by engaging physicians and scientists involved in various aspects of clinical management.

Two Candida auris Cases in Germany with No Recent Contact to Foreign Healthcare-Epidemiological and Microbiological Investigations.

Candida auris has become a global fungal public health threat. This multidrug-resistant yeast is associated with nosocomial intra- and interhospital transmissions causing healthcare-associated infections. Here, we report on two C. auris cases from Germany. The two patients stayed in Germany for a long time before C. auris was detected during their hospitalization. The patients were isolated in single rooms with contact precautions. No nosocomial transmissions were detected within the hospital. Both C. auris isolates exhibited high minimum inhibitory concentrations (MICs) of fluconazole and one isolate additionally high MICs against the echinocandins. Microsatellite genotyping showed that both strains belong to the South Asian clade. These two cases are examples for appropriate in-hospital care and infection control without further nosocomial spread. Awareness for this emerging, multidrug-resistant pathogen is justified and systematic surveillance in European health care facilities should be performed.

Next-Generation Sequencing-Based Decision Support for Intensivists in Difficult-to-Diagnose Disease States: A Case Report of Invasive Cerebral Aspergillosis.

Aspergillus spp. are widespread environmental pathogens that can induce invasive aspergillosis, especially in immunocompromised patients. An 86-year-old female patient presented with a rare case of invasive cerebral aspergillosis. The aspergilloma invaded the intracranial region originating from the ethmoidal sinus and the orbital apex. In contrast to routine diagnostic procedures, next-generation sequencing (NGS) was able to identify the fungal pathogen in the cerebrospinal fluid as well as in plasma samples, supporting the biopsy-based diagnosis of invasive cerebral aspergillosis. Therefore, NGS-based diagnostics may be of particular importance for difficult-to-diagnose disease states, when conventional diagnostic procedures fail.

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from two German hospitals.

Introduction: Vancomycin-resistant Enterococcus faecium accounts for around 10-23% of nosocomial enterococcal infections and constitutes a relevant therapeutic problem due to its limited susceptibility to antibiotics. The resistance towards glycopeptide antibiotics is mediated by the so-called van genes. Currently, the most common resistance type in Germany is the vanB-type. Little data are available on the molecular epidemiology in Germany. Therefore, an epidemiological typing of Enterococcus faecium isolates with vanB-type resistance from two German hospitals in Essen and Nuremberg was performed. Two outbreaks and 104 sporadic cases were investigated. Methods: All 128 isolates with vanB-type resistance were collected between 2011-2012 and 2017-2018. They were characterized using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results: ST 117 was the most common sequence type (ST) in both hospitals, especially since 2017. PFGE divided the isolates of this study into 68 PFGE types and showed a broad genetic diversity. Two epidemiologically assumed in-hospital outbreaks were genetically confirmed. Apart from that, in-hospital transmissions were rare events. Conclusion: The results obtained by MLST confirmed the previously described allocation of STs in Germany. PFGE showed a broad genetic diversity of vanB VRE between the two hospitals and also within each hospital. In-hospital transmissions were rare, but outbreaks did occur. Our data supports the strategy to screen and isolate patients in transmission events in order to detect monoclonality indicating a common source or hygiene mismanagement.