Allele-specific collateral and fitness effects determine the dynamics of fluoroquinolone resistance evolution.

Collateral sensitivity (CS), which arises when resistance to one antibiotic increases sensitivity toward other antibiotics, offers treatment opportunities to constrain or reverse the evolution of antibiotic resistance. The applicability of CS-informed treatments remains uncertain, in part because we lack an understanding of the generality of CS effects for different resistance mutations, singly or in combination. Here, we address this issue in the gram-positive pathogen Streptococcus pneumoniae by measuring collateral and fitness effects of clinically relevant gyrA and parC alleles and their combinations that confer resistance to fluoroquinolones. We integrated these results in a mathematical model that allowed us to evaluate how different in silico combination treatments impact the dynamics of resistance evolution. We identified common and conserved CS effects of different gyrA and parC alleles; however, the spectrum of collateral effects was unique for each allele or allelic pair. This indicated that allelic identity can impact the evolutionary dynamics of resistance evolution during monotreatment and combination treatment. Our model simulations, which included the experimentally derived antibiotic susceptibilities and fitness effects, and antibiotic-specific pharmacodynamics revealed that both collateral and fitness effects impact the population dynamics of resistance evolution. Overall, we provide evidence that allelic identity and interactions can have a pronounced impact on collateral effects to different antibiotics and suggest that these need to be considered in models examining CS-based therapies.

Evolution of genome fragility enables microbial division of labor.

Division of labor can evolve when social groups benefit from the functional specialization of its members. Recently, a novel means of coordinating the division of labor was found in the antibiotic-producing bacterium Streptomyces coelicolor, where specialized cells are generated through large-scale genomic re-organization. We investigate how the evolution of a genome architecture enables such mutation-driven division of labor, using a multiscale computational model of bacterial evolution. In this model, bacterial behavior-antibiotic production or replication-is determined by the structure and composition of their genome, which encodes antibiotics, growth-promoting genes, and fragile genomic loci that can induce chromosomal deletions. We find that a genomic organization evolves, which partitions growth-promoting genes and antibiotic-coding genes into distinct parts of the genome, separated by fragile genomic loci. Mutations caused by these fragile sites mostly delete growth-promoting genes, generating sterile, and antibiotic-producing mutants from weakly-producing progenitors, in agreement with experimental observations. This division of labor enhances the competition between colonies by promoting antibiotic diversity. These results show that genomic organization can co-evolve with genomic instabilities to enable reproductive division of labor.

Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System.

Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.

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.

Gut Microbiota Colonization and Transmission in the Burying Beetle Nicrophorus vespilloides throughout Development.

Carrion beetles in the genus Nicrophorus rear their offspring on decomposing carcasses where larvae are exposed to a diverse community of decomposer bacteria. Parents coat the carcass with antimicrobial secretions prior to egg hatch (defined as prehatch care) and also feed regurgitated food, and potentially bacteria, to larvae throughout development (defined as full care). Here, we partition the roles of prehatch and posthatch parental care in the transmission and persistence of culturable symbiotic bacteria to larvae. Using three treatment groups (full care, prehatch care only, and no care), we found that larvae receiving full care are predominantly colonized by bacteria resident in the maternal gut while larvae receiving no care are colonized with bacteria from the carcass. More importantly, larvae receiving only prehatch care were also predominantly colonized by maternal bacteria; this result indicates that parental treatment of the carcass, including application of bacteria to the carcass surface, is sufficient to ensure symbiont transfer even in the absence of direct larval feeding. Later in development, we found striking evidence that pupae undergo an aposymbiotic stage, after which they are recolonized at eclosion with bacteria similar to those found on the molted larval cuticle and on the wall of the pupal chamber. Our results clarify the importance of prehatch parental care for symbiont transmission in Nicrophorus vespilloides and suggest that these bacteria successfully outcompete decomposer bacteria during larval and pupal gut colonization.IMPORTANCE Here, we examine the origin and persistence of the culturable gut microbiota of larvae in the burying beetle Nicrophorus vespilloides This insect is particularly interesting for this study because larvae are reared on decomposing vertebrate carcasses, where they are exposed to high densities of carrion-decomposing microbes. Larvae also receive extensive parental care in the form of carcass preservation and direct larval feeding. We find that parents transmit their gut bacteria to larvae both directly, through regurgitation, and indirectly via their effects on the carcass. In addition, we find that larvae become aposymbiotic during pupation but are recolonized apparently from bacteria shed onto the insect cuticle before adult eclosion. Our results highlight the diverse interactions between insect behavior and development on microbiota composition. They further suggest that competitive interactions mediate the bacterial composition of Nicrophorus larvae together with or apart from the influence of beetle immunity, suggesting that the bacterial communities of these insects may be highly coevolved with those of their host species.

Conservative sex and the benefits of transformation in Streptococcus pneumoniae.

Natural transformation has significant effects on bacterial genome evolution, but the evolutionary factors maintaining this mode of bacterial sex remain uncertain. Transformation is hypothesized to have both positive and negative evolutionary effects on bacteria. It can facilitate adaptation by combining beneficial mutations into a single individual, or reduce the mutational load by exposing deleterious alleles to natural selection. Alternatively, it may expose transformed cells to damaged or otherwise mutated environmental DNA and is energetically expensive. Here, we examine the long-term effects of transformation in the naturally competent species Streptococcus pneumoniae by evolving populations of wild-type and competence-deficient strains in chemostats for 1000 generations. Half of these populations were exposed to periodic mild stress to examine context-dependent benefits of transformation. We find that competence reduces fitness gain under benign conditions; however, these costs are reduced in the presence of periodic stress. Using whole genome re-sequencing, we show that competent populations fix fewer new mutations and that competence prevents the emergence of mutators. Our results show that during evolution in benign conditions competence helps maintain genome stability but is evolutionary costly; however, during periods of stress this same conservativism enables cells to retain fitness in the face of new mutations, showing for the first time that the benefits of transformation are context dependent.

Egg survival is reduced by grave-soil microbes in the carrion beetle, Nicrophorus vespilloides.

BACKGROUND: Nicrophorus vespilloides eggs are deposited into the soil in close proximity to the decomposing vertebrate carcasses that these insects use as an obligate resource to rear their offspring. Eggs in this environment potentially face significant risks from the bacteria that proliferate in the grave-soil environment following nutrient influx from the decomposing carcass. Our aims in this paper are twofold: first, to examine the fitness effects of grave-soil bacteria to eggs, and second, to quantify egg immunocompetence as a defence against these bacteria. RESULTS: Our results provide strong evidence that grave-soil microbes significantly reduce the survival of Nicrophorus eggs. Females provided with microbe rich carcasses to rear broods laid fewer eggs that were less likely to hatch than females given uncontaminated carcasses. Furthermore, we show that egg hatch success is significantly reduced by bacterial exposure. Using a split-brood design, which controlled for intrinsic differences in eggs produced by different females, we found that eggs washed free of surface-associated bacteria show increased survival compared to unwashed eggs. By contrast, eggs exposed to the entomopathogen Serratia marcescens show decreased survival compared to unexposed eggs. We next tested the immune competence of eggs under challenge from bacterial infection, and found that eggs lacked endogenous production of antimicrobial peptides, despite well-developed responses in larvae. Finally, we found that despite lacking immunity, N. vespilloides eggs produce an extraembryonic serosa, indicating that the serosa has lost its immune inducing capacity in this species. CONCLUSIONS: The dependency on ephemeral resources might strongly select for fast developing animals. Our results suggest that Nicrophorus carrion beetles, and other species developing on ephemeral resources, face a fundamental trade-off between egg immunity and development time.

Phenotypic heterogeneity in Streptomyces colonies.

Streptomyces are a large genus of multicellular bacteria best known for their prolific production of bioactive natural products. In addition, they play key roles in the mineralisation of insoluble resources, such as chitin and cellulose. Because of their multicellular mode of growth, colonies of interconnected hyphae extend over a large area that may experience different conditions in different parts of the colony. Here, we argue that within-colony phenotypic heterogeneity can allow colonies to simultaneously respond to divergent inputs from resources or competitors that are spatially and temporally dynamic. We discuss causal drivers of heterogeneity, including competitors, precursor availability, metabolic diversity and division of labour, that facilitate divergent phenotypes within Streptomyces colonies. We discuss the adaptive causes and consequences of within-colony heterogeneity, highlight current knowledge (gaps) and outline key questions for future studies.

Using click chemistry to study microbial ecology and evolution.

Technological advances have largely driven the revolution in our understanding of the structure and function of microbial communities. Culturing, long the primary tool to probe microbial life, was supplanted by sequencing and other -omics approaches, which allowed detailed quantitative insights into species composition, metabolic potential, transcriptional activity, secretory responses and more. Although the ability to characterize "who's there" has never been easier or cheaper, it remains technically challenging and expensive to understand what the diverse species and strains that comprise microbial communities are doing in situ, and how these behaviors change through time. Our aim in this brief review is to introduce a developing toolkit based on click chemistry that can accelerate and reduce the expense of functional analyses of the ecology and evolution of microbial communities. After first outlining the history of technological development in this field, we will discuss key applications to date using diverse labels, including BONCAT, and then end with a selective (biased) view of areas where click-chemistry and BONCAT-based approaches stand to have a significant impact on our understanding of microbial communities.

Towards evolutionary predictions: Current promises and challenges.

Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS-CoV2 and influenza to CRISPR-based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions.

Variation in Streptococcus pneumoniae susceptibility to human antimicrobial peptides may mediate intraspecific competition.

Streptococcus pneumoniae is a facultative pathogen inhabiting the nasopharynx of humans where it is exposed to a range of antimicrobial peptides (AMPs) of the innate immune response. It is possible therefore that the susceptibility of strains to AMPs plays a role in determining their ability to colonize, and furthermore, that AMPs could mediate competitive interactions between co-colonizing genotypes. However, little is known about patterns of natural variation in AMP susceptibility of S. pneumoniae, and it is unclear whether the susceptibilities of an isolate to multiple human AMPs are correlated. We tested this by characterizing the susceptibility of 31 S. pneumoniae natural isolates to human neutrophil peptide (HNP-1) (α-defensin) and LL-37 (cathelicidin). We observed significant variation in susceptibility between isolates to both AMPs, and in the majority of isolates, susceptibilities to HNP-1 and LL-37 were uncorrelated. Clinical isolates were more susceptible to AMPs than were carriage isolates. The polysaccharide capsule of S. pneumoniae is thought to protect cells against AMPs. However, serotype alone could not explain the observed variation in susceptibility suggesting that genetic background plays an equally important role. We tested directly whether AMPs could mediate competition between isolates using competition experiments in the presence and absence of AMPs. These experiments demonstrated that AMPs could indeed reverse the outcome of competition between selected isolates. AMP-mediated competition could therefore contribute to the maintenance of intraspecific genetic diversity in S. pneumoniae.

Killing as means of promoting biodiversity.

Bacteriocins are usually viewed as the effective weapons of bacterial killers. However, killing competitors with bacteriocins may be not only a means of eliminating other strains, but also a crucial unappreciated mechanism promoting bacterial diversity. In the present short review, we summarize recent empirical and theoretical studies examining the role bacteriocins that may play in driving and maintaining diversity among microbes. We conclude by highlighting limitations of current models and suggest directions for future studies.

Ecological drivers of division of labour in Streptomyces.

Division of labour occurs when different individuals, cells or tissues become specialised to perform complementary tasks that benefit the whole organism or social group. Although long studied in multicellular organisms and colonies of social insects, several recent studies have established that division of labour is common in microorganisms. We review recent work on the division of labour in unicellular and multicellular bacteria, with a particular focus on reproductive and metabolic divisions of labour in actinomycetes. Actinomycetes show enormous variation in sporophore morphology and spore production patterns that likely affect the potential for cooperative interactions within colonies. They also display both irreversible genetic and spatiotemporally regulated phenotypic divisions of labour that structure antibiotic production. We highlight outstanding questions in this group of multicellular bacteria and outline factors that can modify the expression of division of labour across microbes.

Mutational meltdown of putative microbial altruists in Streptomyces coelicolor colonies.

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a subpopulation of cells arises that hyperproduces metabolically costly antibiotics, resulting in a division of labor that increases colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is similar to altruistic worker castes in social insects or somatic cells in multicellular organisms. To understand these mutant cells' reproductive and genomic fate after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that in contrast to wild-type cells, putatively altruistic mutant cells continue to decline in fitness during transfer while they lose more fragments from their chromosome ends. In addition, the base-substitution rate in mutants increases roughly 10-fold, possibly due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with DNA damage due to point mutations and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest the cells arising in the S. coelicolor division of labor are analogous to altruistic reproductively sterile castes of social insects.

Polymorphic competence peptides do not restrict recombination in Streptococcus pneumoniae.

Understanding the factors that limit recombination in bacteria is critical in order to better understand and assess its effects on genetic variation and bacterial population genetic structure. Transformation in the naturally competent bacterium, Streptococcus pneumoniae, is regulated by a polymorphic competence (com) apparatus. It has been suggested that polymorphic types, called pherotypes, generate and maintain subpopulation genetic structure within this species. We test predictions stemming from this hypothesis using a cosmopolitan sample of clinical pneumococcal isolates. We sequenced the locus encoding the peptide that induces competence (comC) to assign clones to each known pherotype class and then used multilocus sequence typing to determine whether there is significant genetic differentiation between pherotypes subgroups. We find two dominant pherotypes within our sample, and both are maintained at high frequencies (CSP1 74%, CSP2 26%). Our analyses fail to detect significant genetic differentiation between pherotype groups and find strong evidence, from a coalescent analysis, for interpherotype recombination. In addition, our analyses indicate that positive selection may account for the maintenance of the fixed polymorphism in this locus (comC). Altogether, these results fail to support the prediction that the polymorphism in the competence system acts to limit recombination within S. pneumoniae populations. We discuss why this result is expected given the mechanism underlying transformation and outline a scenario to explain the evolution of polymorphism in the competence system.

Next-generation sequencing as a tool to study microbial evolution.

Thanks to their short generation times and large population sizes, microbes evolve rapidly. Evolutionary biologists have exploited this to observe evolution in real time. The falling costs of whole-genome sequencing using next-generation technologies now mean that it is realistic to use this as a tool to study this rapid microbial evolution both in the laboratory and in the wild. Such experiments are being used to accurately estimate the rates of mutation, reveal the genetic targets and dynamics of natural selection, uncover the correlation (or lack thereof) between genetic and phenotypic change, and provide data to test long-standing evolutionary hypotheses. These advances have important implications for our understanding of the within- and between-host evolution of microbial pathogens.

Design principles of collateral sensitivity-based dosing strategies.

Collateral sensitivity (CS)-based antibiotic treatments, where increased resistance to one antibiotic leads to increased sensitivity to a second antibiotic, may have the potential to limit the emergence of antimicrobial resistance. However, it remains unclear how to best design CS-based treatment schedules. To address this problem, we use mathematical modelling to study the effects of pathogen- and drug-specific characteristics for different treatment designs on bacterial population dynamics and resistance evolution. We confirm that simultaneous and one-day cycling treatments could supress resistance in the presence of CS. We show that the efficacy of CS-based cycling therapies depends critically on the order of drug administration. Finally, we find that reciprocal CS is not essential to suppress resistance, a result that significantly broadens treatment options given the ubiquity of one-way CS in pathogens. Overall, our analyses identify key design principles of CS-based treatment strategies and provide guidance to develop treatment schedules to suppress resistance.

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.

Signal diffusion and the mitigation of social exploitation in pneumococcal competence signalling.

Quorum sensing (QS) in bacteria is thought to enable populations of cells to coordinately and cooperatively regulate gene expression for traits that confer group benefits. While this view has strong empirical and theoretical support, it is increasingly appreciated that QS under natural conditions may be incapable of monitoring bacterial numbers and, furthermore, that QS is evolutionarily unstable owing to conflicts of interest among competing cells. An alternative hypothesis, termed diffusion sensing (DS), proposes that autoinducer secretion monitors the diffusive properties of the local environment, with benefits that are directly realized by individual cells rather than populations. Here, we test central predictions of this hypothesis using the competence signalling system of Streptococcus pneumoniae as our model, which regulates the induction of natural transformation by the secretion and detection of a small diffusible peptide, CSP (competence-stimulating peptide). By experimentally manipulating the diffusive properties of the growth medium, we found that there is no fixed quorum for competence induction. Instead, induction cell density scales with diffusivity. In agreement with QS and DS expectations, we show that the benefit of signal exploitation by mutant cells that can use but not secrete CSP is strongly frequency-dependent. However, we also find that the magnitude of this benefit declines significantly as diffusion is reduced, a result more consistent with the predictions of DS. Together, these data provide strong support for the DS hypothesis for autoinducer response systems. More specifically, our results imply that autonomous rather than group benefits should be sought in order to more completely understand the role and evolution of CSP signalling in pneumococci.