Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated “S-Protein-Typer” tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad C_t range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12–13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.

Objectives. This study aimed to evaluate and compare the performance of different assays for antimicrobial susceptibility testing (AST) and minimum inhibitory concentration (MIC) determination for reserve antibiotics in carbapenem-resistant Enterobacterales (CREs), Pseudomonas aeruginosa (CRPAs), and Acinetobacter baumannii (CRABs). Methods. An analysis was conducted on 100 consecutive isolates: 50 CREs, 35 CRPAs, and 15 CRABs. Sensititre broth microdilution was used as a reference standard to evaluate the performance of VITEK 2 card AST-XN24 (bioMérieux), the respective gradient tests (bioMérieux), and UMIC colistin broth microdilution test strips (Bruker Daltonics). Errors, essential agreement (EA), and categorical agreement of MICs for colistin (COL), meropenem–vaborbactam (MVB), imipenem–relebactam (IRL), and eravacycline (ERV) were assessed. Results. The agreement between both of the COL broth microdilution (BMD) methods was perfect (100/100). The gradient test and VITEK 2 analysis yielded comparable EA rates (92/100 and 72/79, respectively), with the latter not registering any very major errors (VMEs). The MVB gradient test achieved EA in 66 of 85 isolates and VITEK 2 in 70/85. For IRL, EA was reached in 69 and 64 of 85 cases by gradient test and VITEK 2 analysis, respectively. The ERV gradient test yielded false results in nearly all (12/15) CRABs but achieved EA in 46 of 50 CREs. The VITEK system recorded EA for ERV in 60 of 65 isolates. Conclusions. We observed substantial variability in the measured MICs between BMD and the alternative methods. In only a few constellations, VITEK 2 or gradient tests could substitute the reference method. BMD is the method of choice for COL analysis, with VITEK 2 representing an alternative method for CRPA testing. Alternative methods for MVB did not provide reliable results, except for Enterobacterales, when tested with the gradient test. However, resistant results need to be confirmed by BMD. Only BMD can be used for IRL MIC determination. VITEK 2 was mostly accurate in measuring ERV MICs, while the corresponding gradient test yielded reliable results exclusively in CREs. It is essential that laboratories are aware of which testing method provides reliable results for each combination of microorganisms and reserve antibiotics.

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.

Objectives: The fourth-generation fluoroquinolone delafloxacin (DFX) is propagated as a novel (oral) treatment option for infections with multidrug-resistant Gram-negatives. To date, efficacy data for Enterobacterales is only available for wild-type isolates but not for those with pre-existing resistance against third-generation fluoroquinolones. Methods: One hundred twenty-nine non-duplicate clinical isolates of Enterobacterales that demonstrated ciprofloxacin resistance in VITEK 2 testing were collected and analyzed for CIP and DFX minimum inhibitory concentrations (MICs) via broth microdilution. Results: DFX MICs ranged from 0.5 to ≥ 16 mg L⁻¹ (median: 4 mg L⁻¹). All Escherichia coli isolates (n = 56; 43% of all isolates), the only species with EUCAST breakpoints, were classified as DFX resistant. All other isolates belonging to species for which EUCAST epidemiological cut-offs (ECOFFs) are available (n = 64; 50% of all isolates) demonstrated MICs above the ECOFF. With a minimum and median MIC of 2 and 8 mg L⁻¹, respectively, the remaining isolates (6%) achieved similar results. A moderate correlation between the MIC values of the two fluoroquinolones was found. For the majority of isolates (64%), other oral options for antibiotic therapy, such as amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole, were still available. Conclusions: Our data suggests that, compared to CIP, DFX does not demonstrate increased in vitro efficacy against fluoroquinolone-resistant Enterobacterales. Subsequent DFX testing seems to be of limited additional value.