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Abstract Fully defined laboratory media have the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. We developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg + K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the physiology of Saccharomyces cerevisiae in different actual molasses-based media. We demonstrate the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. Here we present in detail the development of a fully defined synthetic molasses medium and the physiology of yeast strains in this medium compared to industrial molasses. This tailor-made medium was able to satisfactorily reproduce the physiology of S. cerevisiae in industrial molasses. Thus, we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

Abstract Mouse models are commonly used to study the colonisation profiles of microorganisms introduced to the gastrointestinal tract. Three commonly used mouse models include conventional, germ-free, and antibiotic-treated mice. However, colonisation resistance in conventional mice and specialised equipment for germ-free mice are usually limiting factors in their applications. In this study, we sought to establish a robust colonisation model for Saccharomyces boulardii, a probiotic yeast that has caught attention in the field of probiotics and advanced microbiome therapeutics. We characterised the colonisation of S. boulardii in conventional mice and mice treated with a cocktail of broad-spectrum antibiotics, including ampicillin, kanamycin, metronidazole and vancomycin. We found colonisation levels increased up to 10,000-fold in the antibiotic-treated mice compared to nonantibiotic-treated mice. Furthermore, S. boulardii was detected continuously in more than 75% of mice for 10 days after the last administration in antibiotic-treated mice, in contrast to in nonantibiotic-treated mice where S. boulardii was undetectable in less than 2 days. Finally, we demonstrated that this antibiotic cocktail can be used in two commonly used mouse strains, C57BL/6 and ob/ob mice, both achieving ~ 108 CFU/g of S. boulardii in faeces. These findings highlight that the antibiotic cocktail used in this study is an advantageous tool to study S. boulardii based probiotic and advanced microbiome therapeutics.

Abstract In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

Abstract Adaptive laboratory evolution is an important tool to evolve organisms to increased tolerance towards different physical and chemical stress. It is applied to study the evolution of antibiotic resistance as well as genetic mechanisms underlying improvements in production strains. Adaptive evolution experiments can be automated in a high-throughput fashion. However, the characterization of the resulting lineages can become a time consuming task, when the performance of each lineage is evaluated individually. Here, we present a novel method for the markerless insertion of randomized genetic barcodes into the genome of Escherichia coli using a novel dual-auxotrophic selection approach. The barcoded E. coli library allows multiplexed phenotyping of evolved strains in pooled competition experiments. We use the barcoded library in an adaptive evolution experiment; evolving resistance towards three common antibiotics. Comparing this multiplexed phenotyping with conventional susceptibility testing and growth-rate measurements we can show a significant positive correlation between the two approaches. Use of barcoded bacterial strain libraries for individual adaptive evolution experiments drastically reduces the workload of characterizing the resulting phenotypes and enables prioritization of lineages for in-depth characterization. In addition, barcoded clones open up new ways to profile community dynamics or to track lineages in vivo or situ.