Due to the widespread occurrence of multidrug resistant microbes there is increasing interest in the use of novel nanostructured materials as antimicrobials. Specifically, metallic nanoparticles such as silver, copper, and gold have been deployed due to the multiple impacts they have on bacterial physiology. From this, many have concluded that such nanomaterials represent steep obstacles against the evolution of resistance. However, we have already shown that this view is fallacious. For this reason, the significance of our initial experiments are beginning to be recognized in the antimicrobial effects of nanomaterials literature. This recognition is not yet fully understood and here we further explain why nanomaterials research requires a more nuanced understanding of core microbial evolution principles.

There has been an increased usage of metallic antimicrobial materials to control pathogenic and multi-drug resistant bacteria. Yet, there is a corresponding need to know if this usage leads to genetic adaptations that could produce more harmful strains. Experimental evolution was used to adapt Escherichia coli K-12 MG1655 to excess iron (II) with subsequent genomic analysis. Phenotypic assays and gene expression studies were conducted to demonstrate pleiotropic effects associated with this adaptation and to elucidate potential cellular responses. After 200 days of adaptation, populations cultured in excess iron (II), showed a significant increase in 24-h optical densities compared to controls. Furthermore, these populations showed increased resistance toward other metals [iron (III) and gallium (III)] and to traditional antibiotics (bacitracin, rifampin, chloramphenicol and sulfanilamide). Genomic analysis identified selective sweeps in three genes; fecA, ptsP and ilvG unique to the iron (II) resistant populations, and gene expression studies demonstrated that their cellular response may be to downregulate genes involved in iron transport (cirA and fecA) while increasing the oxidative stress response (oxyR, soxS and soxR) prior to FeSO₄ exposure. Together, this indicates that the selected populations can quickly adapt to stressful levels of iron (II). This study is unique in that it demonstrates that E. coli can adapt to environments that contain excess levels of an essential micronutrient while also demonstrating the genomic foundations of the response and the pleiotropic consequences. The fact that adaptation to excess iron also causes increases in general antibiotic resistance is a serious concern. Lay summary: The evolution of iron resistance in E. coli leads to multi-drug and general metal resistance through the acquisition of mutations in three genes (fecA, ptsP and ilvG) while also initiating cellular defenses as part of their normal growth process.

The rise in antimicrobial resistant bacteria have prompted the need for antibiotic alternatives. To address this problem, significant attention has been given to the antimicrobial use and novel applications of copper. As novel applications of antimicrobial copper increase, it is important to investigate how bacteria may adapt to copper over time. Here, we used experimental evolution with re-sequencing (EER-seq) and RNA-sequencing to study the evolution of copper resistance in Escherichia coli. Subsequently, we tested whether copper resistance led to rifampicin, chloramphenicol, bacitracin, and/or sulfonamide resistance. Our results demonstrate that E. coli is capable of rapidly evolving resistance to CuSO₄ after 37 days of selection. We also identified multiple de novo mutations and differential gene expression patterns associated with copper, most notably those mutations identified in the cpx gene. Furthermore, we found that the copper resistant bacteria had decreased sensitivity when compared to the ancestors in the presence of chloramphenicol, bacitracin, and sulfonamide. Our data suggest that the selection of copper resistance may inhibit growth in the antimicrobials tested, resulting in evolutionary trade-offs. The results of our study may have important implications as we consider the antimicrobial use of copper and how bacteria may respond to increased use over time.

The increasing prevalence of antibiotic-resistant bacteria has necessitated the exploration of alternative antimicrobial agents, particularly natural products like essential oils. This study investigated the antibacterial potential of a unique blend of four essential oils (EOB) across a gradient of concentrations (0.1 to 50%) against Gram-positive and Gram-negative bacteria using an adapted broth microdilution method, minimum inhibitory concentrations (MICs), and 24-h growth assays. The Gram-positive bacteria were Staphylococcus epidermidis and Bacillus subtilis, while the Gram-negative bacteria were Escherichia coli and Klebsiella aerogenes. The results demonstrated that the EOB exerted a concentration-dependent inhibitory effect on bacterial growth, with MICs determined at 25% for all the species tested. Growth curve analysis revealed that lower concentrations of the EOB (0.1 to 0.78%) allowed for normal bacterial proliferation, while at intermediate concentrations (1.56 to 3.13%), inconsistent trends in growth were exhibited. At higher concentrations (25 and 50%), the EOB effectively halted bacterial growth, as indicated by flat growth curves. The increase in the lag phase and the decrease in the growth rate at a sub-MIC concentration (12.5%) suggest a significant effect on bacterial adaptation and survival. Relative fitness analyses further highlighted the inhibitory effects of higher essential oil concentrations. S. epidermidis and E. coli had a significant (p < 0.05) reduction in fitness starting from the 6.25% concentration, while the other two species experienced a significant (p < 0.001) reduction in relative fitness from a concentration of 12.5%. These findings underscore the potential of this EOB as an effective antimicrobial agent, particularly in the context of rising antibiotic resistance. Furthermore, the study suggests that the EOB used in the present study could be integrated into therapeutic strategies as a natural alternative or adjunct to traditional antibiotics, offering a promising avenue for combating resistant bacterial strains.

Long-term space missions have shown an increased incidence of oral disease in astronauts’ and as a result, are one of the top conditions predicted to impact future missions. Here we set out to evaluate the adaptive response of Streptococcus mutans (etiological agent of dental caries) to simulated microgravity. This organism has been well studied on earth and treatment strategies are more predictable. Despite this, we are unsure how the bacterium will respond to the environmental stressors in space. We used experimental evolution for 100-days in high aspect ratio vessels followed by whole genome resequencing to evaluate this adaptive response. Our data shows that planktonic S. mutans did evolve variants in three genes (pknB, SMU_399 and SMU_1307c) that can be uniquely attributed to simulated microgravity populations. In addition, collection of data at multiple time points showed mutations in three additional genes (SMU_399, ptsH and rex) that were detected earlier in simulated microgravity populations than in the normal gravity controls, many of which are consistent with other studies. Comparison of virulence-related phenotypes between biological replicates from simulated microgravity and control orientation cultures generally showed few changes in antibiotic susceptibility, while acid tolerance and adhesion varied significantly between biological replicates and decreased as compared to the ancestral populations. Most importantly, our data shows the importance of a parallel normal gravity control, sequencing at multiple time points and the use of biological replicates for appropriate analysis of adaptation in simulated microgravity.

PORTMAGE recombineering is a simple technique for incorporation of novel point mutations into bacterial genomes that eliminates off-target effects. Here we inserted point mutations into the cusS gene from Escherichia coli, then, using Illumina sequencing, report genetic variants in all mutant strains. Several off-site mutations were found at high frequency. Low frequency mutations also show high heterogeneity. This means that it is essential for studies to report all off-target effects and acknowledge the effect that this may have on resultant phenotypes.