Objectives: The identification and characterization of clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility. Methods: Agar dilution assays were used to determine fidaxomicin MICs. Genome sequence data were obtained by single-molecule real-time (SMRT) sequencing in addition to amplicon sequencing of rpoB and rpoC alleles. Allelic exchange was used to introduce the identified mutation into C. difficile 630Δerm. Replication rates, toxin A/B production and spore formation were determined from the strain with reduced fidaxomicin susceptibility. Results: Out of 50 clinical C. difficile isolates, isolate Goe-91 revealed markedly reduced fidaxomicin susceptibility (MIC >64 mg/L). A V1143D mutation was identified in rpoB of Goe-91. When introduced into C. difficile 630Δerm, this mutation decreased fidaxomicin susceptibility (MIC >64 mg/L), but was also associated with a reduced replication rate, low toxin A/B production and markedly reduced spore formation. In contrast, Goe-91, although also reduced in toxin production, showed normal growth rates and only moderately reduced spore formation capacities. This indicates that the rpoBV1143D allele-associated fitness defect is less pronounced in the clinical isolate. Conclusions: To the best of our knowledge, this is the first description of a pathogenic clinical C. difficile isolate with markedly reduced fidaxomicin susceptibility. The lower-than-expected fitness burden of the resistance-mediating rpoBV1143D allele might be an indication for compensatory mechanisms that take place during in vivo selection of mutants.

Due to the beginning of vaccination against COVID-19, serological discrimination between vaccine-associated humoral response and serology-based surveillance of natural SARS-CoV-2 infections as well as breakthrough infections becomes an issue of relevance. Here, we assessed the differentiated effects of the application of an RNA vaccine using SARS-CoV-2 spike protein epitopes on the results of both anti-spike protein–based serology (EUROIMMUN) and anti-nucleocapsid-based serology (VIROTECH). A total of 80 serum samples from vaccinees acquired at different time points after vaccination was assessed. While positive or borderline serological response in the anti-spike protein assay was observed for all samples (90% both IgG and IgA, 6.3% IgA only, 3.8% borderline IgG only), only a single case of a falsely positive IgM was observed for the anti-nucleocapsid assay as expected due to this assay’s specificity. Positive anti-spike protein antibodies were already detectable in the second week after the first dose of vaccination, with higher titers after the second dose of the vaccine. In conclusion, the combined application of anti-spike protein–based serology and anti-nucleocapsid-based serology will provide a useful option for the discrimination of vaccination response and natural infection.

This study was performed as a head-to-head comparison of the performance characteristics of (1) two SARS-CoV-2-specific rapid antigen assays with real-time PCR as gold standard as well as (2) a fully automated high-throughput transcription-mediated amplification (TMA) assay and real-time PCR in a latent class analysis-based test comparison without a gold standard with several hundred samples in a low prevalence “real world” setting. Recorded sensitivity and specificity of the NADAL and the LumiraDx antigen assays and the Hologic Aptima SARS-CoV-2 TMA assay were 0.1429 (0.0194, 0.5835), 0.7644 (0.7016, 0.8174), and 0.7157 (0, 1) as well as 0.4545 (0.2022, 0.7326), 0.9954 (0.9817, 0.9988), and 0.9997 (not estimable), respectively. Agreement kappa between the positive results of the two antigen-based assays was 0.060 (0.002, 0.167) and 0.659 (0.492, 0.825) for TMA and real-time PCR. Samples with low viral load as indicated by cycle threshold (Ct) values > 30 were generally missed by both antigen assays, while 1:10 pooling suggested higher sensitivity of TMA compared to real-time PCR. In conclusion, both sensitivity and specificity speak in favor of the use of the LumiraDx rather than the NADAL antigen assay, while TMA results are comparably as accurate as PCR, when applied in a low prevalence setting.

Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance genes in Enterobacterales. In line with this, we investigated the plasmid-resistome of seven bla_OXA-48 gene-carrying Klebsiella pneumoniae isolates, which were isolated between 2013 and 2014 at the University Medical Center in Göttingen, Germany. All isolates were subjected to complete genome sequencing including the reconstruction of entire plasmid sequences. In addition, phenotypic resistance testing was conducted. The seven isolates comprised both disease-associated isolates and colonizers isolated from five patients. They fell into two clusters of three sequence type (ST)101 and two ST11 isolates, respectively; and ST15 and ST23 singletons. The seven isolates harbored various plasmids of the incompatibility (Inc) groups IncF, IncL/M, IncN, IncR, and a novel plasmid chimera. All bla_OXA-48 genes were encoded on the IncL/M plasmids. Of note, distinct phenotypical resistance patterns associated with different sets of resistance genes encoded by IncL/M and IncR plasmids were observed among isolates of the ST101 cluster in spite of high phylogenetic relatedness of the bacterial chromosomes, suggesting nosocomial transmission. This highlights the importance of plasmid uptake and plasmid recombination events for the fast generation of resistance variability after clonal transmission. In conclusion, this study contributes a piece in the puzzle of molecular epidemiology of resistance gene-carrying plasmids in K. pneumoniae in Germany.

Serological assays can contribute to the estimation of population proportions with previous immunologically relevant contact with the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) virus. In this study, we compared five commercially available diagnostic assays for the diagnostic identification of SARS-CoV-2-specific antibodies. Depending on the assessed immunoglobulin subclass, recorded sensitivity ranged from 17.0% to 81.9% with best results for immunoglobulin G. Specificity with blood donor sera ranged from 90.2% to 100%, with sera from EBV patients it ranged from 84.3% to 100%. Agreement from fair to nearly perfect was recorded depending on the immunoglobulin class between the assays, the with best results being found for immunoglobulin G. Only for this immunoglobulin class was the association between later sample acquisition times (about three weeks after first positive PCR results) and positive serological results in COVID-19 patients confirmed. In conclusion, acceptable and comparable reliability for the assessed immunoglobulin G-specific assays could be shown, while there is still room for improvement regarding the reliability of the assays targeting the other immunoglobulin classes.

Background: Motility in bacteria forms the basis for taxis and is in some pathogenic bacteria important for virulence. Video tracking of motile bacteria allows the monitoring of bacterial swimming behaviour and taxis on the level of individual cells, which is a prerequisite to study the underlying molecular mechanisms. Results: The open-source python program YSMR (Your Software for Motility Recognition) was designed to simultaneously track a large number of bacterial cells on standard computers from video files in various formats. In order to cope with the high number of tracked objects, we use a simple detection and tracking approach based on grey-value and position, followed by stringent selection against suspicious data points. The generated data can be used for statistical analyses either directly with YSMR or with external programs. Conclusion: In contrast to existing video tracking software, which either requires expensive computer hardware or only tracks a limited number of bacteria for a few seconds, YSMR is an open-source program which allows the 2-D tracking of several hundred objects over at least 5 minutes on standard computer hardware. The code is freely available at https://github.com/schwanbeck/YSMR