Antagonism between Bacteriostatic and Bactericidal Antibiotics Is Prevalent

Combination therapy is rarely used to counter the evolution of resistance in bacterial infections. Expansion of the use of combination therapy requires knowledge of how drugs interact at inhibitory concentrations. More than 50 years ago, it was noted that, if bactericidal drugs are most potent with actively dividing cells, then the inhibition of growth induced by a bacteriostatic drug should result in an overall reduction of efficacy when the drug is used in combination with a bactericidal drug. Our goal here was to investigate this hypothesis systematically. We first constructed time-kill curves using five different antibiotics at clinically relevant concentrations, and we observed antagonism between bactericidal and bacteriostatic drugs. We extended our investigation by performing a screen of pairwise combinations of 21 different antibiotics at subinhibitory concentrations, and we found that strong antagonistic interactions were enriched significantly among combinations of bacteriostatic and bactericidal drugs. Finally, since our hypothesis relies on phenotypic effects produced by different drug classes, we recreated these experiments in a microfluidic device and performed time-lapse microscopy to directly observe and quantify the growth and division of individual cells with controlled antibiotic concentrations. While our single-cell observations supported the antagonism between bacteriostatic and bactericidal drugs, they revealed an unexpected variety of cellular responses to antagonistic drug combinations, suggesting that multiple mechanisms underlie the interactions

The Cost of Virulence: Retarded Growth of Salmonella Typhimurium Cells Expressing Type III Secretion System 1

Virulence factors generally enhance a pathogen's fitness and thereby foster transmission. However, most studies of pathogen fitness have been performed by averaging the phenotypes over large populations. Here, we have analyzed the fitness costs of virulence factor expression by Salmonella enterica subspecies I serovar Typhimurium in simple culture experiments. The type III secretion system ttss-1, a cardinal virulence factor for eliciting Salmonella diarrhea, is expressed by just a fraction of the S. Typhimurium population, yielding a mixture of cells that either express ttss-1 (TTSS-1+ phenotype) or not (TTSS-1− phenotype). Here, we studied in vitro the TTSS-1+ phenotype at the single cell level using fluorescent protein reporters. The regulator hilA controlled the fraction of TTSS-1+ individuals and their ttss-1 expression level. Strikingly, cells of the TTSS-1+ phenotype grew slower than cells of the TTSS-1− phenotype. The growth retardation was at least partially attributable to the expression of TTSS-1 effector and/or translocon proteins. In spite of this growth penalty, the TTSS-1+ subpopulation increased from <10% to approx. 60% during the late logarithmic growth phase of an LB batch culture. This was attributable to an increasing initiation rate of ttss-1 expression, in response to environmental cues accumulating during this growth phase, as shown by experimental data and mathematical modeling. Finally, hilA and hilD mutants, which form only fast-growing TTSS-1− cells, outcompeted wild type S. Typhimurium in mixed cultures. Our data demonstrated that virulence factor expression imposes a growth penalty in a non-host environment. This raises important questions about compensating mechanisms during host infection which ensure successful propagation of the genotype

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting &gt;100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved

Division of labour in response to host oxidative burst drives a fatal Cryptococcus gattii outbreak

Cryptococcus gattii is an emerging intracellular pathogen and the cause of the largest primary outbreak of a life-threatening fungal disease in a healthy population. Outbreak strains share a unique mitochondrial gene expression profile and an increased ability to tubularize their mitochondria within host macrophages. However, the underlying mechanism that causes this lineage of C. gattii to be virulent in immunocompetent individuals remains unexplained. Here we show that a subpopulation of intracellular C. gattii adopts a tubular mitochondrial morphology in response to host reactive oxygen species. These fungal cells then facilitate the rapid growth of neighbouring C. gattii cells with non-tubular mitochondria, allowing for effective establishment of the pathogen within a macrophage intracellular niche. Thus, host reactive oxygen species, an essential component of the innate immune response, act as major signalling molecules to trigger a ‘division of labour’ in the intracellular fungal population, leading to increased pathogenesis within this outbreak lineage

Evolution of stress response in the face of unreliable environmental signals.

Most organisms live in ever-changing environments, and have to cope with a range of different conditions. Often, the set of biological traits that are needed to grow, reproduce, and survive varies between conditions. As a consequence, organisms have evolved sensory systems to detect environmental signals, and to modify the expression of biological traits in response. However, there are limits to the ability of such plastic responses to cope with changing environments. Sometimes, environmental shifts might occur suddenly, and without preceding signals, so that organisms might not have time to react. Other times, signals might be unreliable, causing organisms to prepare themselves for changes that then do not occur. Here, we focus on such unreliable signals that indicate the onset of adverse conditions. We use analytical and individual-based models to investigate the evolution of simple rules that organisms use to decide whether or not to switch to a protective state. We find evolutionary transitions towards organisms that use a combination of random switching and switching in response to the signal. We also observe that, in spatially heterogeneous environments, selection on the switching strategy depends on the composition of the population, and on population size. These results are in line with recent experiments that showed that many unicellular organisms can attain different phenotypic states in a probabilistic manner, and lead to testable predictions about how this could help organisms cope with unreliable signals

Fibronectin fibers are highly tensed in healthy organs in contrast to tumors and virus-infected lymph nodes

The extracellular matrix (ECM) acts as reservoir for a plethora of growth factors and cytokines some of which are hypothesized to be regulated by ECM fiber tension. Yet, ECM fiber tension has never been mapped in healthy versus diseased organs. Using our recently developed tension nanoprobe derived from the bacterial adhesin FnBPA5, which preferentially binds to structurally relaxed fibronectin fibers, we discovered here that fibronectin fibers are kept under high tension in selected healthy mouse organs. In contrast, tumor tissues and virus-infected lymph nodes exhibited a significantly higher content of relaxed or proteolytically cleaved fibronectin fibers. This demonstrates for the first time that the tension of ECM fibers is significantly reduced upon pathological tissue transformations. This has wide implications, as the active stretching of fibronectin fibers adjusts critical cellular niche parameters and thereby tunes the reciprocal cell-ECM crosstalk. Mapping the tensional state of fibronectin fibers opens novel and unexpected diagnostic opportunities.ISSN:2590-028

Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice

The microbiota confers colonization resistance, which blocks Salmonella gut colonization1. As diet affects microbiota composition, we studied whether food composition shifts enhance susceptibility to infection. Shifting mice to diets with reduced fibre or elevated fat content for 24 h boosted Salmonella Typhimurium or Escherichia coli gut colonization and plasmid transfer. Here, we studied the effect of dietary fat. Colonization resistance was restored within 48 h of return to maintenance diet. Salmonella gut colonization was also boosted by two oral doses of oleic acid or bile salts. These pathogen blooms required Salmonella’s AcrAB/TolC-dependent bile resistance. Our data indicate that fat-elicited bile promoted Salmonella gut colonization. Both E. coli and Salmonella show much higher bile resistance than the microbiota. Correspondingly, competitive E. coli can be protective in the fat-challenged gut. Diet shifts and fat-elicited bile promote S. Typhimurium gut infections in mice lacking E. coli in their microbiota. This mouse model may be useful for studying pathogen–microbiota–host interactions, the protective effect of E. coli, to analyse the spread of resistance plasmids and assess the impact of food components on the infection process