Antibiotic resistance begets more resistance: chromosomal resistance mutations mitigate fitness costs conferred by multi-resistant clinical plasmids.

Plasmids are the primary vectors of horizontal transfer of antibiotic resistance genes among bacteria. Previous studies have shown that the spread and maintenance of plasmids among bacterial populations depend on the genetic makeup of both the plasmid and the host bacterium. Antibiotic resistance can also be acquired through mutations in the bacterial chromosome, which not only confer resistance but also result in changes in bacterial physiology and typically a reduction in fitness. However, it is unclear whether chromosomal resistance mutations affect the interaction between plasmids and the host bacteria. To address this question, we introduced 13 clinical plasmids into a susceptible Escherichia coli strain and three different congenic mutants that were resistant to nitrofurantoin (ΔnfsAB), ciprofloxacin (gyrA, S83L), and streptomycin (rpsL, K42N) and determined how the plasmids affected the exponential growth rates of the host in glucose minimal media. We find that though plasmids confer costs on the susceptible strains, those costs are fully mitigated in the three resistant mutants. In several cases, this results in a competitive advantage of the resistant strains over the susceptible strain when both carry the same plasmid and are grown in the absence of antibiotics. Our results suggest that bacteria carrying chromosomal mutations for antibiotic resistance could be a better reservoir for resistance plasmids, thereby driving the evolution of multi-drug resistance.IMPORTANCEPlasmids have led to the rampant spread of antibiotic resistance genes globally. Plasmids often carry antibiotic resistance genes and other genes needed for its maintenance and spread, which typically confer a fitness cost on the host cell observed as a reduced growth rate. Resistance is also acquired via chromosomal mutations, and similar to plasmids they also reduce bacterial fitness. However, we do not know whether resistance mutations affect the bacterial ability to carry plasmids. Here, we introduced 13 multi-resistant clinical plasmids into a susceptible and three different resistant E. coli strains and found that most of these plasmids do confer fitness cost on susceptible cells, but these costs disappear in the resistant strains which often lead to fitness advantage for the resistant strains in the absence of antibiotic selection. Our results imply that already resistant bacteria are a more favorable reservoir for multi-resistant plasmids, promoting the ascendance of multi-resistant bacteria.

Staphylococcus aureus mutants resistant to the feed-additive monensin show increased virulence and altered purine metabolism.

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

Vesicle-enriched secretomes alter bacterial competitive abilities and are drivers of evolution in microbial communities.

Microbial membrane vesicles can carry compounds that inhibit bacterial growth, but how they impact the fitness of the vesicle-producing bacterial species and influence community dynamics remain unexplored questions. To address these questions, we examined the effect of vesicle-enriched secretomes (VESs) in different single-species and multi-species systems. Effects of VESs on single-species growth dynamics were determined for nine bacterial species belonging to four genera (Escherichia, Salmonella, Pseudomonas and Bacillus) in nutrient-rich and poor growth media. Results showed both species-specific and nutrient-dependent effects of the VESs on bacterial growth. The strongest antagonistic effects were observed for VES isolated from the natural isolates of E. coli, while those isolated from P. aeruginosa PA14 affected the highest number of species. We further demonstrated that these VESs altered the competitive abilities of the species involved in two-species (S. Typhimurium LT2 and S. arizonae) and three-species systems (E. coli, S. Typhimurium LT2 and B. subtilis). Finally, using experimental evolution we showed that different bacterial species could rapidly acquire mutations that abrogated the antagonistic effects of VESs. This study demonstrates how VESs can contribute in shaping microbial communities, both by increasing the competitive ability of a given bacterial species and as a driver of genetic adaptation.

Pervasive Selection for Clinically Relevant Resistance and Media Adaptive Mutations at Very Low Antibiotic Concentrations.

Experimental evolution studies have shown that weak antibiotic selective pressures (i.e., when the antibiotic concentrations are far below the minimum inhibitory concentration, MIC) can select resistant mutants, raising several unanswered questions. First, what are the lowest antibiotic concentrations at which selection for de novo resistance mutations can occur? Second, with weak antibiotic selections, which other types of adaptive mutations unrelated to the antibiotic selective pressure are concurrently enriched? Third, are the mutations selected under laboratory settings at subMIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at subMICs in the presence of either of four clinically used antibiotics: fosfomycin, nitrofurantoin, tetracycline, and ciprofloxacin. Antibiotic resistance evolution was investigated at concentrations ranging from 1/4th to 1/2000th of the MIC of the susceptible strain (MICsusceptible). Our results show that evolution was rapid across all the antibiotics tested, and selection for fosfomycin- and nitrofurantoin-resistant mutants was observed at a concentration as low as 1/2000th of MICsusceptible. Several of the evolved resistant mutants showed increased growth yield and exponential growth rates, and outcompeted the susceptible ancestral strain in the absence of antibiotics as well, suggesting that adaptation to the growth environment occurred in parallel with the selection for resistance. Genomic analysis of the resistant mutants showed that several of the mutations selected under these conditions are also found in clinical isolates, demonstrating that experimental evolution at very low antibiotic levels can help in identifying novel mutations that contribute to bacterial adaptation during subMIC exposure in real-life settings.

Different adaptive strategies in E. coli populations evolving under macronutrient limitation and metal ion limitation.

BACKGROUND: Adaptive responses to nutrient limitation involve mutations that increase the efficiency of usage or uptake of the limiting nutrient. However, starvation of different nutrients has contrasting effects on physiology, resulting in different evolutionary responses. Most studies performed to understand these evolutionary responses have focused only on macronutrient limitation. Hence our understanding of adaptation under limitation of other forms of nutrients is limited. In this study, we compared the evolutionary response in populations evolving under growth-limiting conditions for a macronutrient and a major cation. RESULTS: We evolved eight populations of E. coli in nutrient-limited chemostats for 400 generations to identify the genetic basis of the mechanisms involved in efficient usage of two nutrients: nitrogen and magnesium. Our population genomic sequencing work, based on this study and previous work, allowed us to identify targets of selection under these nutrient limiting conditions. Global transcriptional regulators glnGL were targets of selection under nitrogen starvation, while proteins involved in outer-membrane biogenesis (genes from the lpt operon) were targets of selection under magnesium starvation. The protein involved in cell-cycle arrest (yhaV) was a target of selection in both environments. We re-constructed specific mutants to analyze the effect of individual mutations on fitness in nutrient limiting conditions in chemostats and in batch cultures. We further demonstrated that adaptation to nitrogen starvation proceeds via a nutrient specific mechanism, while that to magnesium starvation involves a more general mechanism. CONCLUSIONS: Our results show two different forms of adaptive strategies under limitation of nutrients that effect cellular physiology in different ways. Adaptation to nitrogen starvation proceeds by upregulation of transcriptional regulator glnG and subsequently of transporter protein amtB, both of which results in increased nitrogen scavenging ability of the cell. On the other hand, adaptation to magnesium starvation proceeds via the restructuring of the cell outer-membrane, allowing magnesium to be redistributed to other biological processes. Also, adaptation to the chemostat environment involves selection for loss of function mutations in genes that under nutrient-limiting conditions interfere with continuous growth.

Evolutionary implications of Liebig's law of the minimum: Selection under low concentrations of two nonsubstitutable nutrients.

Interactions between different axes of an organism's niche determine the evolutionary trajectory of a population. An extreme case of these interactions is predicted from ecological theory in Liebig's law of the minimum. This law states that in environments where multiple nutrients are in relatively low concentrations, only one nutrient will affect the growth of the organism. This implies that the evolutionary response of the population would be dictated by the most growth-limiting nutrient. Alternatively, it is possible that an initial adaptation to the most limiting nutrient results in other nutrients present in low concentration affecting the evolutionary dynamics of the population. To test these hypotheses, we conducted twelve evolution experiments in chemostats using Escherichia coli populations: four under nitrogen limitation, four under magnesium limitation, and four in which both nitrogen and magnesium are in low concentrations. In the last environment, only magnesium seems to limit growth (Low Nitrogen Magnesium Limited environment, LNML). We observe a decrease in nitrogen concentration in the LNML environment over the course of our evolution experiment indicating that nitrogen might become limiting in these environments. Genetic reconstruction results show that clones adapted to magnesium limitation have genes involved in nitrogen starvation, that is, glnG (nitrogen starvation transcriptional regulator) and amtB (transport protein) to be upregulated only in the LNML environment as compared to magnesium-limiting environments. Together, our results highlights that in low-nutrient environments, adaptation to the growth-limiting nutrient results in other nutrients at low concentrations to play a role in the evolutionary dynamics of the population.

Bats (Chiroptera: Noctilionoidea) Challenge a Recent Origin of Extant Neotropical Diversity.

The mechanisms underlying the high extant biodiversity in the Neotropics have been controversial since the 19th century. Support for the influence of period-specific changes on diversification often rests on detecting more speciation events during a particular period. The timing of speciation events may reflect the influence of incomplete taxon sampling, protracted speciation, and null processes of lineage accumulation. Here we assess the influence of these factors on the timing of speciation with new multilocus data for New World noctilionoid bats (Chiroptera: Noctilionoidea). Biogeographic analyses revealed the importance of the Neotropics in noctilionoid diversification, and the critical role of dispersal. We detected no shift in speciation rate associated with the Quaternary or pre-Quaternary periods, and instead found an increase in speciation linked to the evolution of the subfamily Stenodermatinae (∼18 Ma). Simulations modeling constant speciation and extinction rates for the phylogeny systematically showed more speciation events in the Quaternary. Since recording more divergence events in the Quaternary can result from lineage accumulation, the age of extant sister species cannot be interpreted as supporting higher speciation rates during this period. Instead, analyzing the factors that influence speciation requires modeling lineage-specific traits and environmental, spatial, and ecological drivers of speciation.

From the field to the lab: best practices for field preservation of bat specimens for molecular analyses.

Studies in molecular ecology depend on field-collected samples for genetic information, and the tissue sampled and preservation conditions strongly affect the quality of the DNA obtained. DNA yields from different tissue types have seldom been compared, and the relative performance of storage media has never been directly tested, even though these media may influence DNA degradation under field conditions. We analyzed DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as quantitative PCR for a single-copy nuclear locus. We also compared DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant. Wing punches yielded more total DNA than did buccal swabs, and wing tissues preserved in silica beads yielded significantly more total and nuclear DNA than those preserved in DMSO or ethanol. These results show that tissue type and preservation media strongly influence the quantity of DNA obtained from non-lethal genetic samples, and based on these effects we provide recommendations for field collection of tissues for genetic analyses.

Integrating incomplete fossils by isolating conflicting signal in saturated and non-independent morphological characters.

Morphological characters are indispensable in phylogenetic analyses for understanding the pattern, process, and tempo of evolution. If characters are independent and free of systematic errors, then combining as many different kinds of characters as are available will result in the best-supported phylogenetic hypotheses. But since morphological characters are subject to natural selection for function and arise from the expression of developmental pathways, they may not be independent, a situation that may amplify any underlying homoplasy. Here, we use new dental and multi-locus genetic data from bats (Mammalia: Chiroptera) to quantify saturation and similarity in morphological characters and introduce two likelihood-based approaches to identify strongly conflicting characters and integrate morphological and molecular data. We implement these methods to analyze the phylogeny of incomplete Miocene fossils in the radiation of Phyllostomidae (New World Leaf-nosed Bats), perhaps the most ecologically diverse family of living mammals. Morphological characters produced trees incongruent with molecular phylogenies, were saturated, and showed rates of change higher than most molecular substitution rates. Dental characters encoded variation similar to that in other morphological characters, while molecular characters encoded highly dissimilar variation in comparison. Saturation and high rates of change indicate randomization of phylogenetic signal in the morphological data, and extensive similarity suggests characters are non-independent and errors are amplified. To integrate the morphological data into tree building while accounting for homoplasy, we used statistical molecular scaffolds and combined phylogenetic analyses excluding a small subset of strongly conflicting dental characters. The phylogenies revealed the Miocene nectar-feeding †Palynephyllum nests within the crown nectar-feeding South American subfamily Lonchophyllinae, while the Miocene genus †Notonycteris is sister to the extant carnivorous Vampyrum. These relationships imply new calibration points for timing of radiation of the ecologically diverse Phyllostomidae. [Chiroptera; conflict; dentition; morphology; Phyllostomidae; saturation; scaffold; systematic error.].

Selection for mechanical advantage underlies multiple cranial optima in new world leaf-nosed bats.

Selection for divergent performance optima has been proposed as a central mechanism underlying adaptive radiation. Uncovering multiple optima requires identifying forms associated with different adaptive zones and linking those forms to performance. However, testing and modeling the performance of complex morphologies like the cranium is challenging. We introduce a three-dimensional finite-element (FE) model of the cranium that can be morphed into different shapes by varying simple parameters to investigate the relationship between two engineering-based measures of performance, mechanical advantage and von Mises stress, and four divergent adaptive zones occupied by New World Leaf-nosed bats. To investigate these relationships, we tested the fit of Brownian motion and Ornstein-Uhlenbeck models of evolution in mechanical advantage and von Mises stress using dated multilocus phylogenies. The analyses revealed three performance optima for mechanical advantage among species from three adaptive zones: bats that eat nectar; generalized insectivores, omnivores and some frugivores; and bats that specialize on hard canopy fruits. Only two optima, one corresponding to nectar feeding, were consistently uncovered for von Mises stress. These results suggest that mechanical advantage played a larger role than von Mises stress in the radiation of New World Leaf-nosed bats into divergent adaptive zones.