Within-species variability of antibiotic interactions in Gram-negative bacteria.

Treatments with antibiotic combinations are becoming increasingly important even though the supposed clinical benefits of combinations are, in many cases, unclear. Here, we systematically examined how several clinically used antibiotics interact and affect the antimicrobial efficacy against five especially problematic Gram-negative pathogens. A total of 232 bacterial isolates were tested against different pairwise antibiotic combinations spanning five classes, and the ability of all combinations in inhibiting growth was quantified. Descriptive statistics, principal component analysis (PCA), and Spearman's rank correlation matrix were used to determine the correlations between the different combinations on interaction outcome. Several important conclusions can be drawn from the 696 examined interactions. Firstly, within a species, the interactions are in general conserved but can be isolate-specific for a given antibiotic combination and can range from antagonistic to synergistic. Secondly, additive and antagonistic interactions are the most common observed across species and antibiotics, with 87.1% of isolate-antibiotic combinations being additive, 11.6% antagonistic, and only 0.3% showing synergy. These findings suggest that to achieve the highest precision and efficacy of combination therapy, not only isolate-specific interaction profiling ought to be routinely performed, in particular to avoid using drug combinations that show antagonistic interaction and an expected associated reduction in efficacy, but also discovering rare and potentially valuable synergistic interactions.IMPORTANCEAntibiotic combinations are often used to treat bacterial infections, which aim to increase treatment efficacy and reduce resistance evolution. Typically, it is assumed that one specific antibiotic combination has the same effect on different isolates of the same species, i.e., the interaction is conserved. Here, we tested this idea by examining how several clinically used antibiotics interact and affect the antimicrobial efficacy against several bacterial pathogens. Our results show that, even though within a species the interactions are often conserved, there are also isolate-specific differences for a given antibiotic combination that can range from antagonistic to synergistic. These findings suggest that isolate-specific interaction profiling ought to be performed in clinical microbiology routine to avoid using antagonistic drug combinations that might reduce treatment efficacy.

Competition Sensing Changes Antibiotic Production in Streptomyces.

One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require "elicitors" to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors, and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing strains of streptomycetes, we show that strains commonly inhibit each other's growth and that this occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreases in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competition. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes.IMPORTANCE Bacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces cells are more likely to produce antibiotics when grown with competitors that are closely related or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor's toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces cells also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

Spatial structure increases the benefits of antibiotic production in Streptomyces.

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.

Distance-dependent danger responses in bacteria.

The last decade has seen a resurgence in our understanding of the diverse mechanisms that bacteria use to kill one another. We are also beginning to uncover the responses and countermeasures that bacteria use when faced with specific threats or general cues of potential danger from bacterial competitors. In this Perspective, we propose that diverse offensive and defensive responses in bacteria have evolved to offset dangers detected at different distances. Thus, while volatile organic compounds provide bacterial cells with a warning at the greatest distance, diffusible compounds like antibiotics or contact mediated killing systems, indicate a more pressing danger warranting highly-specific responses. In the competitive environments in which bacteria live, it is crucial that cells are able to detect real or potential dangers from other cells. By utilizing mechanisms of detection that can infer the distance from danger, bacteria can fine-tune aggressive interactions so that they can optimally respond to threats occurring with distinct levels of risk.

The evolution of no-cost resistance at sub-MIC concentrations of streptomycin in Streptomyces coelicolor.

At the high concentrations used in medicine, antibiotics exert strong selection on bacterial populations for the evolution of resistance. However, these lethal concentrations may not be representative of the concentrations bacteria face in soil, a recognition that has led to questions of the role of antibiotics in soil environments as well as the dynamics of resistance evolution during sublethal challenge. Here we examine the evolution of resistance to sub-minimal inhibitory concentrations (sub-MIC) of streptomycin in the filamentous soil bacterium Streptomyces coelicolor. First, we show that spontaneous resistance to streptomycin causes an average fitness deficit of ~21% in the absence of drugs; however, these costs are eliminated at concentrations as low as 1/10 the MIC of susceptible strains. Using experimental evolution, we next show that resistance to >MIC levels of streptomycin readily evolves when bacteria are exposed to sub-MIC doses for 500 generations. Furthermore, the resistant clones that evolved at sub-MIC streptomycin concentrations carry no fitness cost. Whole-genome analyses reveal that evolved resistant clones fixed some of the same mutations as those isolated at high drug concentrations; however, all evolved clones carry additional mutations and some fixed mutations that either compensate for costly resistance or have no associated fitness costs. Our results broaden the conditions under which resistance can evolve in nature and suggest that rather than low-concentration antibiotics acting as signals, resistance evolves in response to antibiotics used as weapons.

Socially mediated induction and suppression of antibiosis during bacterial coexistence.

Despite their importance for humans, there is little consensus on the function of antibiotics in nature for the bacteria that produce them. Classical explanations suggest that bacteria use antibiotics as weapons to kill or inhibit competitors, whereas a recent alternative hypothesis states that antibiotics are signals that coordinate cooperative social interactions between coexisting bacteria. Here we distinguish these hypotheses in the prolific antibiotic-producing genus Streptomyces and provide strong evidence that antibiotics are weapons whose expression is significantly influenced by social and competitive interactions between competing strains. We show that cells induce facultative responses to cues produced by competitors by (i) increasing their own antibiotic production, thereby decreasing costs associated with constitutive synthesis of these expensive products, and (ii) by suppressing antibiotic production in competitors, thereby reducing direct threats to themselves. These results thus show that although antibiotic production is profoundly social, it is emphatically not cooperative. Using computer simulations, we next show that these facultative strategies can facilitate the maintenance of biodiversity in a community context by converting lethal interactions between neighboring colonies to neutral interactions where neither strain excludes the other. Thus, just as bacteriocins can lead to increased diversity via rock-paper-scissors dynamics, so too can antibiotics via elicitation and suppression. Our results reveal that social interactions are crucial for understanding antibiosis and bacterial community dynamics, and highlight the potential of interbacterial interactions for novel drug discovery by eliciting pathways that mediate interference competition.

Adipocyte-derived lipids modulate CD4+ T-cell function.

Adipose tissue contains several immune cells whose number and phenotype vary depending on the adiposity. In the present study, we show that IFN-γ(+) CD4(+) T cells are enriched in human adipose tissue compared with in blood. To gain insight into the underlying mechanisms, we investigated the possibility that human adipocytes modulate CD4(+) T-cell cytokine production and proliferation and show that CD4(+) T cells produced increased levels of IFN-γ when activated in the presence of adipocytes. This effect was mediated by soluble mediators, as shown in transwell and adipocyte-conditioned medium (ACM) transfer experiments. Additionally, ACM induced increased proliferation of CD4(+) T cells upon activation. Intriguingly, the proliferation-enhancing effect resided mainly in the lipid fraction of ACM, as shown upon separation of the protein and lipid fraction. Further separation of these lipids based on polarity revealed that the modulatory effect is confined to fractions containing free fatty acids. All identified fatty acids were able to individually enhance T-cell proliferation. These data indicate that adipocytes can modulate CD4(+) T-cell function through the release of lipids. Remarkably, free fatty acids were the most prominent modulators of T-cell proliferation, possibly leading to an accumulation of these cells in adipose tissue.