Isolation of Persister Cells of <i>Bacillus subtilis</i> and Determination of Their Susceptibility to Antimicrobial Peptides

Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism Bacillus subtilis can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate B. subtilis persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both B. subtilis vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant B. subtilis spores are not susceptible to the antimicrobial peptides

Genomic versus Plasmid-Borne Expression of Germinant Receptor Proteins in <i>Bacillus cereus</i> Strain 14579

Germinant receptors (GRs) are proteins in the spore-forming bacteria of Bacillus species that are crucial in triggering spore germination by sensing nutrients in the spores’ environment. In the Gram-positive bacterium Bacillus cereus strain ATCC 14579, the GerR GR initiates germination with L-alanine. While we have expressed GerR subunits fused to reporter proteins from genes under control of their native promoter on plasmids in this B. cereus strain, here we sought increased flexibility in this work by studying genome integration and plasmid-borne inducible high level (over) expression. However, construction of chromosomal integrants to visualize and localize the GerR B subunit fused to fluorescent reporter protein SGFP2 was not successful in this B. cereus strain using constructs with either shorter (~600 bp) or longer (~1200 bp) regions of homology to the gerR operon. This failure was in contrast to successful IPTG-inducible expression of GerRB-SGFP2 from plasmid pDG148 in vegetative cells and dormant spores, as fluorescent GerRB-SGFP2 foci were present in vegetative cells and the protein was detected by Western blot analysis. In dormant spores, the fluorescence intensity with IPTG-inducible expression from pDG148-gerRB-SGFP2 was significantly higher than in wild type spores. However, the full length GerRB-SGFP2 protein was not detected in spores using Western blots. Clearly, there are still challenges in the construction of B. cereus strains harboring fluorescent reporter proteins in which tagged proteins are encoded by genes incorporated in the chromosome or on extrachromosomal expression plasmids

M2R: a Python add-on to cobrapy for modifying human genome-scale metabolic reconstruction using the gut microbiota models

Motivation: The gut microbiota is the human body's largest population of microorganisms that interact with human intestinal cells. They use ingested nutrients for fundamental biological processes and have important impacts on human physiology, immunity and metabolome in the gastrointestinal tract. Results: Here, we present M2R, a Python add-on to cobrapy that allows incorporating information about the gut microbiota metabolism models to human genome-scale metabolic models (GEMs) like RECON3D. The idea behind the software is to modify the lower bounds of the exchange reactions in the model using aggregated in- and out-fluxes from selected microbes. M2R enables users to quickly and easily modify the pool of the metabolites that enter and leave the GEM, which is particularly important for those looking into an analysis of the metabolic interaction between the gut microbiota and human cells and its dysregulation

Future challenges to microbial food safety

Despite significant efforts by all parties involved, there is still a considerable burden of foodborne illness, in which micro-organisms play a prominent role. Microbes can enter the food chain at different steps, are highly versatile and can adapt to the environment allowing survival, growth and production of toxic compounds. This sets them apart from chemical agents and thus their study from food toxicology. We summarize the discussions of a conference organized by the Dutch Food and Consumer Products Safety Authority and the European Food Safety Authority. The goal of the conference was to discuss new challenges to food safety that are caused by micro-organisms as well as strategies and methodologies to counter these. Management of food safety is based on generally accepted principles of Hazard Analysis Critical Control Points and of Good Manufacturing Practices. However, a more pro-active, science-based approach is required, starting with the ability to predict where problems might arise by applying the risk analysis framework. Developments that may influence food safety in the future occur on different scales (from global to molecular) and in different time frames (from decades to less than a minute). This necessitates development of new risk assessment approaches, taking the impact of different drivers of change into account. We provide an overview of drivers that may affect food safety and their potential impact on foodborne pathogens and human disease risks. We conclude that many drivers may result in increased food safety risks, requiring active governmental policy setting and anticipation by food industries whereas other drivers may decrease food safety risks. Monitoring of contamination in the food chain, combined with surveillance of human illness and epidemiological investigations of outbreaks and sporadic cases continue to be important sources of information. New approaches in human illness surveillance include the use of molecular markers for improved outbreak detection and source attribution, sero-epidemiology and disease burden estimation. Current developments in molecular techniques make it possible to rapidly assemble information on the genome of various isolates of microbial species of concern. Such information can be used to develop new tracking and tracing methods, and to investigate the behavior of micro-organisms under environmentally relevant stress conditions. These novel tools and insight need to be applied to objectives for food safety strategies, as well as to models that predict microbial behavior. In addition, the increasing complexity of the global food systems necessitates improved communication between all parties involved: scientists, risk assessors and risk managers, as well as consumer