Assessing long-term ecological impacts of PCE contamination in groundwater using a flow cytometric fingerprint approach.

This study aims to develop and validate a comprehensive method for assessing ecological disturbances in groundwater ecosystems caused by tetrachloroethylene (PCE) contamination, utilizing flow cytometry (FCM) fingerprint approach. We hypothesized that the ecological disturbance resulting from PCE contamination would exhibit 'press disturbance', persisting over extended periods, and inducing notable phenotypic differences in the microbial community compared to undisturbed groundwater. We collected 40 groundwater samples from industrial district with a history of over twenty years of PCE contamination, along with 56 control groundwater from the national surveillance groundwater system. FCM revealed significant alterations in the phenotypic diversity of microbial communities in PCE-contaminated groundwater, particularly during the dry season. The presence of specific dechlorinating bacteria (Dehalococcoides, Dehalogenimonas, and Geobacter) and their syntrophic partners was identified as an indicator of contamination. Phenotypic diversity measures provided clearer and more direct reflections of contamination impact compared to taxonomic diversity measures. This study establishes FCM fingerprinting as a simple, robust, and accurate method for evaluating ecological disturbances, with potential applications in early warning systems and continuous monitoring of groundwater contamination. The findings not only underscore the sensitivity of FCM in detecting phenotypic variations induced by environmental stressors but also highlight its utility in understanding the complex dynamics of microbial communities in contaminated groundwater ecosystems.

Phenotypic shifts induced by environmental pre-stressors modify antibiotic resistance in Staphylococcus aureus.

Introduction: Escalating prevalence of antibiotic resistance in Staphylococcus aureus has necessitated urgent exploration into the fundamental mechanisms underlying antibiotic resistance emergence, particularly in relation to its interaction with environmental stressors. This study aimed to investigate the effects of environmental stressors prior to antibiotic exposure on the antibiotic resistance of S. aureus. Methods: We used Raman spectroscopy and flow cytometry to measure prior stress-induced phenotypic alterations of S. aureus, and identified the association between phenotypic shifts and the antibiotic resistance. Results: The results revealed a multifaceted relationship between stressors and the development of antibiotic resistance. The stressors effectuate distinct phenotypic diversifications and subsequently amplify these phenotypic alterations following antibiotic treatments, contingent upon the specific mode of action; these phenotypic shifts in turn promote the development of antibiotic resistance in S. aureus. This study's findings demonstrated that the presence of pre-stress conditions triggered an augmentation of resistance to vancomycin (VAN), while concurrently attenuating resistance to norfloxacin. Marked shifts in Raman peaks associated with lipids and nucleic acids demonstrated correlations with elevated survival rates following VAN treatment. Conclusion: Consequently, these observations indicate that pre-stress conditions "prime" bacterial cells for differential responses to antibiotics and bear significant implications for formulating clinical therapeutic strategies.

Transcriptional Response and Plant Growth Promoting Activity of Pseudomonas fluorescens DR397 under Drought Stress Conditions.

Drought is one of the most vulnerable factors that affect crop productivity. Little is known about plant-associated microbiomes and their functional roles in assisting plant growth under drought. We investigated the genetic and transcriptomic characteristics of opportunistic beneficial microorganisms that selectively alleviate stress through plant-bacteria interactions under drought. Pseudomonas fluorescens DR397 was isolated from the drought-prone rhizospheric soil of soybean and showed high metabolic activity at -1.25 Mpa. The genome of DR397 possesses several genes related to the synthesis of compatible solutes (choline and glycine-betaine), exopolysaccharides (alginate and cellulose), and secretion systems (type II, III, IV, and VI), as well as genes related to plant growth promotion (indole-3-acetic acid, transketolase, and thiamine phosphate synthesis). The expression of these genes was significantly upregulated (8- to 263-fold change) only under drought conditions with plant root exudate treatment, whereas subtle transcriptomic changes were observed under solely root exudate treatment. When DR397 was placed on both legume cultivars (Pisum sativum and Phaseolus vulgaris), growth was hardly affected under well-watered conditions, but the shoot and root growths were increased by up from 62.0% to 149.1% compared with the control group under drought conditions. These results provide fundamental insight on the plant-bacterial interactions that alleviate plant stress as an important ecological strategy for improving drought tolerance. IMPORTANCE Drought is a serious abiotic stress on plants as wells as the microbes that coexist with plants, which significantly lowers their fitness. The plant-bacterial interaction is an important strategy to enhance their fitness under drought. However, many knowledge gaps still exist in our understanding of transcriptomic features of bacteria interacting with plant under drought. Here, by investigating the transcriptomic profiles and pot cultivation with legume, we show that the interactions of Pseudomonas fluorescens DR397 with plants change with drought. We, therefore, provide a fundamental evidence of a hidden hero in the soil that promote plant fitness from external stress.

Raman-Deuterium Isotope Probing and Metagenomics Reveal the Drought Tolerance of the Soil Microbiome and Its Promotion of Plant Growth.

Drought has become a major agricultural threat leading crop yield loss. Although a few species of rhizobacteria have the ability to promote plant growth under drought, the drought tolerance of the soil microbiome and its relationship with the promotion of plant growth under drought are scarcely studied. This study aimed to develop a novel approach for assessing drought tolerance in agricultural land by quantitatively measuring microbial phenotypes using stable isotopes and Raman spectroscopy. Raman spectroscopy with deuterium isotope probing was used to identify the Raman signatures of drought effects from drought-tolerant bacteria. Counting drought-tolerant cells by applying these phenotypic properties to agricultural samples revealed that 0% to 52.2% of all measured single cells had drought-tolerant properties, depending on the soil sample. The proportions of drought-tolerant cells in each soil type showed similar tendencies to the numbers of revived pea plants cultivated under drought. The phenotype of the soil microbiome and plant behavior under drought conditions therefore appeared to be highly related. Studying metagenomics suggested that there was a reliable link between the phenotype and genotype of the soil microbiome that could explain mechanisms that promote plant growth in drought. In particular, the proportion of drought-tolerant cells was highly correlated with genes encoding phytohormone production, including tryptophan synthase and isopentenyl-diphosphate delta-isomerase; these enzymes are known to alleviate drought stress. Raman spectroscopy with deuterium isotope probing shows high potential as an alternative technology for quantitatively assessing drought tolerance through phenotypic analysis of the soil microbiome. IMPORTANCE Soil microbiome has played a critical role in the plant survival during drought. However, the drought tolerance of soil microbiome and its ability to promote plant growth under drought is still scarcely studied. In this study, we identified the Raman signature (i.e., phenotype) of drought effects from drought-tolerant bacteria in agricultural soil samples using Raman-deuterium isotope probing (Raman-DIP). Moreover, the number of drought-tolerant cells measured by Raman-DIP was highly related to the survival rate of plant cultivation under drought and the abundance of genes encoding phytohormone production alleviating drought stress in plant. These results suggest Raman-DIP is a promising technology for measuring drought tolerance of soil microbiome. This result give us important insight into further studies of a reliable link between phenotype and genotype of soil microbiome for future plant-bacteria interaction research.

Nutritional status regulates algicidal activity of Aeromonas sp. L23 against cyanobacteria and green algae.

Algicidal bacteria have received broad acceptance as an ecofriendly tool for controlling harmful algal blooms. However, their practical application is still limited to the lab-scale tests due to the complex alga-bacterium interactions in different nutrient statuses. In this study, the Aeromonas sp. L23 that exhibit relatively wide-spectrum in algicidal activity was isolated from a eutrophic agricultural lake. The physiological response of cyanobacteria and green to the algicidal activity under varied nutritional status were studied in an alga-bacterial co-culture. The algicidal activities of L23 against Microcystis aeruginosa UTEX LB 2385, Microcystis aeruginosa NHSB, Anabaena variabilis AG10064, Scenedesmus quadricauda AG10003, and Chlorella vulgaris AG10034 were 88 ± 1.2%, 94 ± 2.6%, 93 ± 0.5%, 82 ± 1.1%, and 47 ± 0.9%, respectively. The L23 cells had low algicidal activity in cell pellet (3%-9%) compared with the cell-free supernatant (78%-93%), indicating that the activity is induced by extracellular substances. Adding glucose, NaNO3, NH4Cl, and KH2PO4 to the co-culture raised the algicidal activity of the L23 against green algae by 5%-50%. Conversely, a 10%-20% decrease in activity occurred against the target cyanobacteria except M. aeruginosa UTEX LB 2385. These results indicated that the interspecific algicidal activity changes according to the nutritional status, which means that the alga-bacterium interaction will be more complex in the field where the nutritional status changes from time to time.