Clostridioides difficile: Analysis on Single Cell Motility and on Antibiotic Resistance
by Julian Schwanbeck
Date of Examination:2020-10-26
Date of issue:2020-12-22
Advisor:Prof. Dr. Uwe Groß
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Fabian Commichau
Referee:Prof. Dr. Rolf Daniel
Referee:Prof. Dr. Burkhard Morgenstern
Referee:PD Dr. Michael Hoppert
Persistent Address:
http://dx.doi.org/10.53846/goediss-8323
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Description:Dissertation: Clostridioides difficile: Analysis on Single Cell Motility and on Antibiotic Resistance
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Abstract
English
Clostridioides difficile, formerly in the genus Clostridium, is a Gram positive obligatory anaerobic spore forming bacterium. C. difficile is the leading nosocomial gastrointestinal pathogen in the western world. In symptomatic patients it causes diarrhoea and in some cases pseudo-membraneous colitis. It can cause toxic megacolon, a potentially lethal complication. After treatment, a large percentage of patients suffer a recurrence. A healthy gastrointestinal microbiome offers some protection against C. difficile infection. Fidaxomicin is an antibiotic effective against C. difficile which has a lesser negative effect on the microbiome compared to other antibiotic regimen. Fidaxomicin resistant C. difficile strains have previously only been created under laboratory conditions. Most mutations conferring decreased susceptibility to Fidaxomicin occur in the RNA polymerase, with the strongest resistance effect caused by mutations in the beta–subunit at amino acid 1143, valine to aspartate (rpoBV1143D). The change in charge of the side chain likely interferes with the binding of Fidaxomicin to the RNA polymerase. Mutations at position 1143 in rpoB come with strong negative side effects, namely reductions in growth rate, sporulation, and toxin formation. We have verified the resistance and side effects of the rpoBV1143D in our lab strain C. difficile 630 erm. In a strain screening we identified a Fidaxomicin resistant C. difficile strain, Goe-91, isolated from a symptomatic patient who was treated with Fidaxomicin. The strain carried a rpoBV1143D mutation, but had no discernible growth rate reduction when compared to our lab strain C. difficile 630 erm. This indicates that Goe-91 has a compensatory mechanism for the negative side effects of the rpoBV1143D mutation, which allow it to remain viable and infectious. Using the C. difficile 630 erm rpoBV1143D strain, we performed an evolutionary experiment selecting for increases in growth rate under Fidaxomicin as selective agent. After a growth period of 16 to 20 days, the resulting three isolates which had a significant increase in growth rate were genome sequenced. Two strains had mutations in the RNA polymerase, proline 244 to threonine in rpoB and glutamate 768 to valine in rpoC. Both mutations likely change how the DNA is guided through the RNA polymerase holoenzyme, which could alleviate the negative effects caused by the rpoBV1143D mutation. As the original Goe-91 strain, as well as one of the three strains from the evolutionary experiment, had no additional mutations in the RNA polymerase, further compensatory mechanisms may exist. The time scale of the evolutionary experiment further fits into an extended pulsed Fidaxomicin regimen, increasing the chance that Fidaxomicin resistant strains will become more common in the future. Most C. difficile strains have an intact motility and chemotaxis operon, but the role of motility and chemotaxis of C. difficile was up to now not fully clear. In order to assess the motility of single cell bacteria on a statistical significant level without human intervention, Y SMR, an open-source python program, was created in this work. It allows for the detection and tracking of several thousand objects on simple desktop PCs and the analysis of speed, percentage of motile cells, stop events, average direction of travel, displacement and run phase length on a population level. Motile C. difficile cells in liquid media display a futile motility phenotype, rapidly moving back and forth in place without achieving displacement. When long chained molecules are added, such as polyvinylpyrrolidone or mucin, it instead performs long run phases, achieving displacement exceeding five cell lengths. When stopping, it inverts its direction of travel and proceeds in a reverse direction. C. difficile has no discernible difference in curvature during run phases regardless of heading, nor a preferred direction of travel. Its motility phenotype therefore does not fit any previously described, and we propose it forms a new phenotype which we termed run and turnaround. The adaption of the motility system of C. difficile to a medium containing long chained or net like molecules is logical, as C. difficile must swim through the mucin layer which protects the epithelial cells. When cysteine is added to the medium at a concentration of 5 mM, C. difficile strains with an intact chemotaxis system react with a slow-down during run phases resulting in a net decrease in displacement. A C. difficile cheY strain in contrast had no change in behaviour. The chemotaxis system in C. difficile therefore works different to that of other described bacteria. Instead of leading to a reorientation of the cell or change in mode of travel, it reduces the rate of rotation of the flagella without affecting the turnaround phases. Cysteine is exported at the membrane of the epithelial cells as part of the gastrointestinal redox system. We propose a motility model for C. difficile in which cysteine serves as a positional indicator which leads to motility reduction and, as a consequence, to an accumulation of cells at their preferred ecological niche.
Keywords: Clostridioides difficile; Antibiotic resistance; Fidaxomicin; Motility; Chemotaxis; RNA Polymerase; Evolution