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The ability to fight bacterial infections with antibiotics has been a longstanding cornerstone of modern medicine. However, wide-spread overuse and misuse of antibiotics has led to unintended consequences, which in turn require large-scale changes of policy for mitigation. In this review, we address two broad classes of corollaries of antibiotics overuse and misuse. Firstly, we discuss the spread of antibiotic resistance from hotspots of resistance evolution to the environment, with special concerns given to potential vectors of resistance transmission. Secondly, we outline the effects of antibiotic pollution independent of resistance evolution on natural microbial populations, as well as invertebrates and vertebrates. We close with an overview of current regional policies tasked with curbing the effects of antibiotics pollution and outline areas in which such policies are still under development.

Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, β-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes.

With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed.

Key Points The fitness effects of antibiotic-resistance mutations and of mutations that compensate for the cost of resistance depends on the molecular basis of resistance and the ecological (or treatment) context in which resistance evolves. The distribution of fitness effects of resistance mutations is determined by antibiotic dose and drug–target interactions. Resistance mutations impose a fitness cost that varies widely among mutations. It may be possible to predict costs of resistance by considering the effects of resistance mutations on protein stability. Compensatory mutations alleviate the cost of resistance, allowing resistant strains to persist in the absence of antibiotics. The opportunity for compensation varies among resistant mutants and it may be possible to predict this variability by explicitly considering the mechanistic basis of the costs of resistance. Physiological interactions between antibiotics and genetic interactions between resistance mutations are crucial for the evolution of multidrug resistance by modifying the benefits associated with resistance and compensatory mutation. Immigration from antibiotic-free populations is important in the evolution of resistance. Immigration accelerates the evolution of resistance when resistance mutations are rare and immigration can reverse resistance following the cessation of antibiotic use. Spatial and temporal patterns of antibiotic use play a key part in the evolution of resistance. Resistance evolves most slowly under maximal levels of environmental heterogeneity. Future work should concentrate on developing predictive models of resistance evolution by integrating molecular mechanisms of resistance with treatment context. This may help develop improved treatment strategies for preventing resistance evolution in pathogen populations. The authors discuss the evolutionary dynamics of antibiotic resistance in bacteria in relation to the complex interplay between population genetic factors and the spatial and temporal pattern of antibiotic use. Despite efforts from a range of disciplines, our ability to predict and combat the evolution of antibiotic resistance in pathogenic bacteria is limited. This is because resistance evolution involves a complex interplay between the specific drug, bacterial genetics and both natural and treatment ecology. Incorporating details of the molecular mechanisms of drug resistance and ecology into evolutionary models has proved useful in predicting the dynamics of resistance evolution. However, putting these models to practical use will require extensive collaboration between mathematicians, molecular biologists, evolutionary ecologists and clinicians.

Pseudomonas aeruginosa is an environmental pathogen that can cause severe infections in immunocompromised patients. P . aeruginosa infections are typically treated with multiple antibiotics including tobramycin, ciprofloxacin, and meropenem. However, antibiotics do not always entirely clear the bacteria from the infection site, where they may remain virulent. This is because the effective antibiotic concentration and diffusion in vitro may differ from the in vivo environment in patients. Therefore, it is important to understand the effect of non-lethal sub-inhibitory antibiotic concentrations on bacterial phenotype. Here, we investigate if sub-inhibitory antimicrobial concentrations cause alterations in bacterial virulence factor production using pyocyanin as a model toxin. We tested this using the aforementioned antibiotics on 10 environmental P . aeruginosa strains. Using on-the-spot electrochemical screening, we were able to directly quantify changes in production of pyocyanin in a measurement time of 17 seconds. Upon selecting 3 representative strains to further test the effects of sub-minimum inhibitory concentration (MICs), we found that pyocyanin production changed significantly when the bacteria were exposed to 10-fold MIC of the 3 antibiotics tested, and this was strain specific. A series of biologically relevant measured pyocyanin concentrations were also used to assess the effects of increased virulence on a culture of epithelial cells. We found a decreased viability of the epithelial cells when incubated with biologically relevant pyocyanin concentrations. This suggests that the antibiotic-induced virulence also is a value worth being enclosed in regular testing of pathogens.

The global spread of antibiotic resistance could be due to a number of factors, and not just the overuse of antibiotics in agriculture and medicine as previously thought.The global spread of antibiotic resistance could be due to a number of factors, and not just the overuse of antibiotics in agriculture and medicine as previously thought.

Wastewater treatment plant (WWTP) discharges alter water quality and microbial communities by introducing human-associated bacteria in the environment and by altering microbial communities. To fully understand this impact, it is crucial to study whether WWTP discharges affect water and sediments microbial communities in comparable ways and whether such effects depend on specific environmental variables. Here, we present a dataset investigating the impact of a WWTP on water quality and bacterial communities by comparing samples collected directly from the WWTP outflow to surface waters and sediments at two sites above and two sites below it over a period of five months. When possible, we measured five physicochemical variables (e.g., temperature, turbidity, conductivity, dissolved oxygen, and salinity), four bioindicators (e.g., Escherichia coli , total coliforms, Enterococcus sp., and endotoxins), and two molecular indicators (e.g., intI1 ’s relative abundance, and 16S rRNA gene profiling). Preliminary results suggest that bioindicators correlate with environmental variables and that bacterial communities present in the water tables, sediments, and treated water differ greatly in composition and structure. Measurement(s) temperature of water • conductivity of water • dissolved oxygen in water • salinity of water • Concentration of Escherichia coli in water • Concentration of total coliforms in water • Concentration of Enterococcus sp. • Concentration of endotoxins in water • Relative abundance of integron 1 in water • Bacterial 16S RNA Technology Type(s) YSI field probe • Colilert dection system • Enterolert dection system • Charles River Endosafe system • quantitative PCR • Illumina Sequencing Sample Characteristic - Organism Bacteria Sample Characteristic - Environment Fresh water river Sample Characteristic - Location United States

Climate-driven shifts in seasonal frost patterns raise important questions about their impact on food quality and resilience. Here, we show that a single 12-h frost event at harvest can enhance both the microbial and nutritional properties of fermented cabbage and carrots, two cold-tolerant crops widely grown in the U.S. Northeast. Using microbial amplicon and metagenomic sequencing, we found that frost exposure led to subtle but consistent changes in microbial composition, including greater abundance of cold-adapted taxa such as Leuconostoc and Debaryomyces . These changes corresponded to increased abundance of genes involved in vitamin biosynthesis, particularly menaquinone (K₂), cobalamin (B₁₂), and threonine pathways. Nutritional assays confirmed higher concentrations of vitamins A and E in frost-conditioned carrot ferments and increased vitamin K₁ in cabbage. Our findings suggest that exposure to seasonal frost can enhance the health-promoting and sensory qualities of fermented vegetables, offering a novel strategy for value-added, climate-resilient food production in temperate regions.

Plant–soil feedbacks (PSFs) drive plant community diversity via interactions between plants and soil microbes. However, we know little about how frequently PSFs affect plants at the seed stage, and the compositional shifts in fungi that accompany PSFs on germination. We conducted a pairwise PSF experiment to test whether seed germination was differentially impacted by conspecific versus heterospecific soils for seven grassland species. We used metagenomics to characterize shifts in fungal community composition in soils conditioned by each plant species. To investigate whether changes in the abundance of certain fungal taxa were associated with multiple PSFs, we assigned taxonomy to soil fungi and identified putative pathogens that were significantly more abundant in soils conditioned by plant species that experienced negative or positive PSFs. We observed negative, positive, and neutral PSFs on seed germination. Although conspecific and heterospecific soils for pairs with significant PSFs contained host‐specialized soil fungal communities, soils with specialized microbial communities did not always lead to PSFs. The identity of host‐specialized pathogens, that is, taxa uniquely present or significantly more abundant in soils conditioned by plant species experiencing negative PSFs, overlapped among plant species, while putative pathogens within a single host plant species differed depending on the identity of the heterospecific plant partner. Finally, the magnitude of feedback on germination was not related to the degree of fungal community differentiation between species pairs involved in negative PSFs. Synthesis. Our findings reveal the potential importance of PSFs at the seed stage. Although plant species developed specialized fungal communities in rhizosphere soil, pathogens were not strictly host‐specific and varied not just between plant species, but according to the identity of plant partner. These results illustrate the complexity of microbe‐mediated interactions between plants at different life stages that next‐generation sequencing can begin to unravel. We tested whether seeds experienced negative or positive feedbacks and monitored changes in soil fungi of seven plant species over time. We found that three plant species experienced negative plant–soil feedbacks on seed germination. All plant species generated distinct fungal communities over time (and relative to each other), but the identity of putative pathogens specialized to particular hosts differed depending on the identity of the heterospecific plant partner.

The unique microbial communities present on fruit surfaces significantly influence the fermentation process and product quality of artisanal cider production, constituting a microbial terroir analogous to that recognized in viticulture. In this study, we investigated the microbial composition and diversity associated with the apple varietals (Empire, Golden Delicious, and Idared) cultivated by two different orchard producers in the Hudson River Valley of New York. Using 16S rRNA and ITS amplicon sequencing, we identified distinct bacterial and fungal communities that varied significantly according to the apple varietal and orchard location. Notably, the orchard was the dominant factor shaping both the bacterial and fungal communities on the apples’ surfaces, with the varietal differences also playing a significant, albeit secondary, role. For example, we found that the bacterial genera Acidophilim sp. and 1174-901-12 sp., as well as the fungus Sporobolmyces patagonicus, were important markers of the orchard in which the apples were cultivated. These microbial signatures persisted into the early stages of cider fermentation, suggesting their potential influence on the cider quality and flavor profile. Our findings underscore the critical importance of the microbial terroir in cider production, and suggest that targeted management practices can leverage regional microbial diversity to enhance the distinctiveness and marketability of artisanal cider products.