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As many gastro-intestinal pathogens, the majority of Clostridioides difficile strains express flagella together with a complete chemotaxis system. The resulting swimming motility is likely contributing to the colonization success of this important pathogen. In contrast to the well investigated general energy metabolism of C. difficile , little is known about the metabolic requirements for maintaining the ion motive force across the membrane, which in turn powers the flagellar motor. We studied here systematically the effect of various amino acids and carbohydrates on the swimming velocity of C. difficile using video microscopy in conjunction with a software based quantification of the swimming speed. Removal of individual amino acids from the medium identified proline and cysteine as the most important amino acids that power swimming motility. Glycine, which is as proline one of the few amino acids that are reduced in Stickland reactions, was not critical for swimming motility. This suggests that the ion motive force that powers the flagellar motor, is critically depending on proline reduction. A maximal and stable swimming motility was achieved with only four compounds, including the amino acids proline, cysteine and isoleucine together with a single, but interchangeable carbohydrate source such as glucose, succinate, mannose, ribose, pyruvate, trehalose, or ethanolamine. We expect that the identified "minimal motility medium" will be useful in future investigations on the flagellar motility and chemotactic behavior in C. difficile , particularly for the unambiguous identification of chemoattractants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schwanbeck, Oehmig, Groß and Bohne.)

Flagellar motility is important for the pathogenesis of many intestinal pathogens, allowing bacteria to move to their preferred ecological niche. Clostridioides difficile is currently the major cause for bacterial health care-associated intestinal infections in the western world. Most clinical strains produce peritrichous flagella and are motile in soft-agar. However, little knowledge exists on the C. difficile swimming behaviour and its regulation at the level of individual cells. We report here on the swimming strategy of C. difficile at the single cell level and its dependency on environmental parameters. A comprehensive analysis of motility parameters from several thousand bacteria was achieved with the aid of a recently developed bacterial tracking programme. C. difficile motility was found to be strongly dependent on the matrix elasticity of the medium. Long run phases of all four motile C. difficile clades were only observed in the presence of high molecular weight molecules such as polyvinylpyrrolidone (PVP) and mucin, which suggests an adaptation of the motility apparatus to the mucin-rich intestinal environment. Increasing mucin or PVP concentrations lead to longer and straighter runs with increased travelled distance per run and fewer turnarounds that result in a higher net displacement of the bacteria. The observed C. difficile swimming pattern under these conditions is characterised by bidirectional, alternating back and forth run phases, interrupted by a short stop without an apparent reorientation or tumbling phase. This motility type was not described before for peritrichous bacteria and is more similar to some previously described polar monotrichous bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schwanbeck, Oehmig, Groß, Zautner and Bohne.)

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.

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.