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Previous work shows that a host's resident microbial community can provide resistance against an invading pathogen. However, this community is continuously changing over time due to adaptive mutations, and how these changes affect the invasion resistance of these communities remains poorly understood. To address this knowledge gap, we used an experimental evolution approach in synthetic communities of Escherichia coli and Salmonella Typhimurium to investigate how the invasion resistance of this community against a bacterium expressing a virulent phenotype, i.e., colicin secretion, changes over time. We show that evolved communities accumulate mutations in genes involved in carbon metabolism and motility, while simultaneously becoming less resistant to invasion. By investigating two-species competitions and generating a three-species competition model, we show that this outcome is dependent on the strength of interspecies interactions. Our study demonstrates how adaptive changes in microbial communities can make them more prone to the detrimental effects of an invading species.

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.

ABSTRACTPlasmids 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.

ABSTRACT 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.

Abstract 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.