A simple protocol for cultivating the bacterivorous soil nematode Caenorhabditis elegans in its natural ecology in the laboratory.

The complex interplay between an animal and its surrounding environment requires constant attentive observation in natural settings. Moreover, how ecological interactions are affected by an animal's genes is difficult to ascertain outside the laboratory. Genetic studies with the bacterivorous nematode Caenorhabditis elegans have elucidated numerous relationships between genes and functions, such as physiology, behaviors, and lifespan. However, these studies use standard laboratory culture that does not reflect C. elegans true ecology. C. elegans is found growing in nature and reproduced in large numbers in soils enriched with rotting fruit or vegetation, a source of abundant and diverse microbes that nourish the thriving populations of nematodes. We developed a simple mesocosm we call soil-fruit-natural-habitat that simulates the natural ecology of C. elegans in the laboratory. Apples were placed on autoclaved potted soils, and after a soil microbial solution was added, the mesocosm was subjected to day-night, temperature, and humidity cycling inside a growth chamber. After a period of apple-rotting, C elegans were added, and the growing worm population was observed. We determined optimal conditions for the growth of C. elegans and then performed an ecological succession experiment observing worm populations every few days. Our data showed that the mesocosm allows abundant growth and reproduction of C. elegans that resembles populations of the nematode found in rotting fruit in nature. Overall, our study presents a simple protocol that allows the cultivation of C. elegans in a natural habitat in the laboratory for a broad group of scientists to study various aspects of animal and microbial ecology.

Microbial interaction-induced siderophore dynamics lead to phenotypic differentiation of Staphylococcus aureus.

This study investigated the impact of microbial interactions on siderophore dynamics and phenotypic differentiation of Staphylococcus aureus under iron-deficient conditions. Optimization of media demonstrated that the glycerol alanine salts medium was best suited for analyzing the dynamics of siderophore production because of its stable production of diverse siderophore types. The effects of pH and iron concentration on siderophore yield revealed a maximum yield at neutral pH and low iron concentration (10 µg). Microbial interaction studies have highlighted variations in siderophore production when different strains (Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli) are co-cultured with S. aureus. Co-culture of S. aureus with P. aeruginosa eliminated siderophore production in S. aureus, while co-culture of S. aureus with E. coli and S. epidermidis produced one or two siderophores, respectively. Raman spectroscopy revealed that microbial interactions and siderophore dynamics play a crucial role in directing the phenotypic differentiation of S. aureus, especially under iron-deficient conditions. Our results suggest that microbial interactions profoundly influence siderophore dynamics and phenotypic differentiation and that the study of these interactions could provide valuable insights for understanding microbial survival strategies in iron-limited environments.

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.

Single-cell phenotypes revealed as a key biomarker in bacterial-fungal interactions: a case study of Staphylococcus and Malassezia.

IMPORTANCE: Evaluating bacterial-fungal interactions is important for understanding ecological functions in a natural habitat. Many studies have defined bacterial-fungal interactions according to changes in growth rates when co-cultivated. However, the current literature lacks detailed studies on phenotypic changes in single cells associated with transcriptomic profiles to understand the bacterial-fungal interactions. In our study, we measured the single-cell phenotypes of bacteria co-cultivated with fungi using Raman spectroscopy with its transcriptomic profiles and determined the consequence of these interactions in detail. This rapid and reliable phenotyping approach has the potential to provide new insights regarding bacterial-fungal interactions.

Bacterial Resuscitation from Starvation-Induced Dormancy Results in Phenotypic Diversity Coupled with Translational Activity Depending on Carbon Substrate Availability.

Dormancy is a survival strategy of stressed bacteria inhabiting a various environment. Frequent dormant-active transitions owing to environmental changes play an important role in functional redundancy. However, a proper understanding of the phenotypic changes in bacteria during these transitions remains to be clarified. In this study, orthogonal approaches, such as electron microscopy, flow cytometry, and Raman spectroscopy, which can evaluate phenotypic heterogeneity at the single-cell level, were used to observe morphological and molecular phenotypic changes in resuscitated cells, and RNA sequencing (RNASeq) was used to determine the genetic characteristics associated with phenotypes. Within 12 h of the resuscitation process, morphological (cell size and shape) and physiological (growth and viability) characteristics as well as molecular phenotypes (cellular components) were found to be recovered to the extent that they were similar to those in active cells. The recovery rate and detailed phenotypic properties of the resuscitated cells differed significantly depending on the type or concentration of carbon sources. RNASeq analysis revealed that genes related to translation were significantly upregulated under all resuscitation conditions. The simpler the carbon source (e.g., glucose), the higher the expression of genes involved in cellular repair, and the more complex the carbon source (e.g., beef extract), the higher the expression of genes associated with increased energy production associated with cellular aerobic respiration. This study of phenotypic plasticity of resuscitated cells provides fundamental insight into understanding the adaptive fine-tuning of the microbiome in response to environmental changes and the functional redundancy resulting from phenotype heterogeneity.

Phenotypic convergence of bacterial adaption to sub-lethal antibiotic treatment.

Microorganisms can adapt quickly to changes in their environment, leading to various phenotypes. The dynamic for phenotypic plasticity caused by environmental variations has not yet been fully investigated. In this study, we analyzed the time-series of phenotypic changes in Staphylococcus cells during adaptive process to antibiotics stresses using flow cytometry and Raman spectroscopy. The nine antibiotics with four different mode of actions were treated in bacterial cells at a sub-lethal concentration to give adaptable stress. Although the growth rate initially varied depending on the type of antibiotic, most samples reached the maximum growth comparable to the control through the short-term adaptation after 24 h. The phenotypic diversity, which showed remarkable changes depending on antibiotic treatment, converged identical to the control over time. In addition, the phenotype with cellular biomolecules converted into a bacterial cell that enhance tolerance to antibiotic stress with increases in cytochrome and lipid. Our findings demonstrated that the convergence into the phenotypes that enhance antibiotic tolerance in a short period when treated with sub-lethal concentrations, and highlight the feasibility of phenotypic approaches in the advanced antibiotic treatment.

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.

Short-Term Legacy Effects of Mercury Contamination on Plant Growth and nifH-Harboring Microbial Community in Rice Paddy Soil.

Methylmercury (MeHg), which is formed in rice paddy soil, exhibits strong neurotoxicity through bioaccumulation in the food chain. A few groups of microorganisms drive both mercury methylation and nitrogen fixation in the rhizosphere. Little is known about how the shifted soil microbial community by Hg contamination affects nitrogen fixation rate and plant growth in paddy soil. Here, we examined how stimulated short-term Hg amendment affects the nitrogen fixing microbial community and influences plant-microbe interactions. Soil was treated with low (0.2 mg/kg) and high (1.1 mg/kg) concentrations of Hg for 4 weeks; then, rice (Oryza sativa) was planted and grown for 12 weeks. The nitrogen-fixation rate and rice growth were measured. The diversity and structure of the microbial community were analyzed by sequencing the nifH gene before and after rice cultivation. Hg treatments significantly decreased the nitrogen fixation rate and dry weight of the rice plants. The structure of the nifH-harboring community was remarkably changed after rice cultivation depending on Hg treatments. Iron- or sulfate-reducing bacteria, including Desulfobacca, Desulfoporosimus, and Geobacter, were observed as legacy response groups; their abundances increased in the soil after Hg treatment. The high abundance of those groups were maintained in control, but the abundance drastically decreased after rice cultivation in the soil treated with Hg, indicating that symbiotic behavior of rice plants changes according to the legacy effects on Hg contamination. These results suggested that Hg contamination can persist in soil microbial communities, affecting their nitrogen-fixation ability and symbiosis with rice plants in paddy soil.

Microbial phenomics linking the phenotype to fonction: The potential of Raman spectroscopy.

Raman spectroscopy is a promising tool for identifying microbial phenotypes based on single cell Raman spectra reflecting cellular biochemical biomolecules. Recent studies using Raman spectroscopy have mainly analyzed phenotypic changes caused by microbial interactions or stress responses (e.g., antibiotics) and evaluated the microbial activity or substrate specificity under a given experimental condition using stable isotopes. Lack of labelling and the nondestructive pretreatment and measurement process of Raman spectroscopy have also aided in the sorting of microbial cells with interesting phenotypes for subsequently conducting physiology experiments through cultivation or genome analysis. In this review, we provide an overview of the principles, advantages, and status of utilization of Raman spectroscopy for studies linking microbial phenotypes and functions. We expect Raman spectroscopy to become a next-generation phenotyping tool that will greatly contribute in enhancing our understanding of microbial functions in natural and engineered systems.