Slimak, Z. C. | bioRxiv Science

bioRxiv2024

Pond Water Microbiome Taxa Profiles and Antibiotic Resistance Genes Associated with Acidity and Tannins

Microbial communities of small freshwater bodies are poorly understood. Four ponds in Knox County, Ohio, were sampled over two years to investigate the relationship between the microbial taxa profiles, antibiotic resistance genes (ARGs), and environmental factors such as pH and tannin concentrations. For each site, microbial communities were collected by filtration and metagenomes were analyzed by short-read sequencing. Taxa profiles were predicted by the Kraken2/Bracken pipelines. Bacterial taxa with high abundance in these ponds included Betaproteobacteria (Polynucleobacter and Methylopumilus) and Actinobacteria (Planktophila, Nanopelagicus, and Mycolicibacterium). One pond, a former quarry with elevated pH, showed high prevalence of Cyanobacteria with a seasonal shift from Synechococcus to Planktothrix in the fall. Planktothrix increase was associated with acidification. ARGs were quantified using the ShortBRED pipeline to detect and quantify hits to a marker set derived from the Comprehensive Antibiotic Resistance Database (CARD). The top two ARGs with the largest marker hits encode components of a Stenotrophomonas drug efflux pump powered by proton-motive force (smeABC) and a mycobacterial global regulator that activates a drug pump and other cell defenses (mtrA). Pump function and global activation of transcription incur large energy expenditures, whose fitness cost may increase at high external pH where the cells proton-motive force is diminished. The smeABC and mtrA prevalence showed a modest correlation with acidifying conditions (low pH and high tannins) which contribute a large transmembrane pH difference to the proton-motive force, thus increasing the cells energy available for pump function and global gene expression. IMPORTANCECompared to rivers and lakes, pond microbial ecosystems are understudied despite close contact with agriculture and recreation. Environmental microbes offer health benefits as well as hazards for human contact. Small water bodies may act as reservoirs for drug-resistant organisms and transfer of antibiotic resistance genes. Yet, the public is rarely aware of the potential for exposure to ARG-carrying organisms in recreational water bodies. Little is known about the capacity for freshwater microbial communities to remediate drug pollution and which biochemical factors may select against antibiotic resistance genes. This study analyzes the bacterial taxa composition and ARG prevalence including possible influence of factors such as pH and tannic acid levels.

microbiology↗

bioRxiv2023

Fitness Tradeoffs of Multidrug Efflux Pumps in Escherichia coli K-12 in Acid or Base, and with Aromatic Phytochemicals

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness tradeoffs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH is important as it contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with CCCP, or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemicals lipophilicity (logP). MdtEF-TolC and EmrAB-TolC each conferred a fitness cost at pH 5.5, but salicylate and benzoate conferred a net positive fitness contribution for the pump. Expression of pump genes was measured by digital PCR. Between pH 5.5 - 8.0, acrA and emrA were upregulated in log phase, whereas mdtE expression was upregulated in transition-to-stationary phase and at pH 5.5 in log phase. Methyl salicylate did not affect pump gene expression, despite selecting against AcrAB-TolC. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without positive section for others. IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate intrinsic drug resistance in our gut microbiome. Intrinsic drug resistance of bacteria is mediated by PMF-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring low-level resistance to antimicrobial agents at first exposure, while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference ({Delta}pH) which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.

microbiology↗