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Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

ABSTRACT: Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.

SUBMITTER: Kramer CE

PROVIDER: S-EPMC4625966 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledThe McMurdo Dry Valleys (MCM) of southern Victoria Land, Antarctica, harbor numerous ice-covered bodies of water that provide year-round liquid water oases for isolated food webs dominated by the microbial loop. Single-cell microbial eukaryotes (protists) occupy major trophic positions within this truncated food web, ranging from primary producers (e.g., chlorophytes, haptophytes, and cryptophytes) to tertiary predators (e.g., ciliates, dinoflagellates, and choanoflagellates). To advance the understanding of MCM protist ecology and the roles of MCM protists in nutrient and energy cycling, we investigated potential metabolic strategies and microbial interactions of key MCM protists isolated from a well-described lake (Lake Bonney). Fluorescence-activated cell sorting (FACS) of enrichment cultures, combined with single amplified genome/amplicon sequencing and fluorescence microscopy, revealed that MCM protists possess diverse potential metabolic capabilities and interactions. Two metabolically distinct bacterial clades (Flavobacteria and Methylobacteriaceae) were independently associated with two key MCM lake microalgae (Isochrysis and Chlamydomonas, respectively). We also report on the discovery of two heterotrophic nanoflagellates belonging to the Stramenopila supergroup, one of which lives as a parasite of Chlamydomonas, a dominate primary producer in the shallow, nutrient-poor layers of the lake.ImportanceSingle-cell eukaryotes called protists play critical roles in the cycling of organic matter in aquatic environments. In the ice-covered lakes of Antarctica, protists play key roles in the aquatic food web, providing the majority of organic carbon to the rest of the food web (photosynthetic protists) and acting as the major consumers at the top of the food web (predatory protists). In this study, we utilized a combination of techniques (microscopy, cell sorting, and genomic analysis) to describe the trophic abilities of Antarctic lake protists and their potential interactions with other microbes. Our work reveals that Antarctic lake protists rely on metabolic versatility for their energy and nutrient requirements in this unique and isolated environment.

Project description:Microbial communities are composed of populations with vastly different abundances and levels of metabolic and replicative activity, ranging from actively metabolizing and dividing to dormant or nonviable. The 16S rRNA/rDNA ratio is an emerging tool for evaluating cell-level metabolic activity independent of abundance. In this study, we used five long-term enriched model anaerobic digestion (AD) communities to investigate community composition, diversity, structure, and in particular activity based on the rRNA/rDNA ratio. We cross-validated the 16S amplicon-based results using two alternative operational taxonomic unit (OTU) formation methods (conventional 97% sequence similarity and 100% sequence similar zero-radius OTUs by UNOISE3) and compared these to metagenome-derived population genomes and metatranscriptomes. Significant positive correlations were observed between microbial total activity and abundance with both the amplicon- and omic-based methods. All three methods revealed disproportionately high transcription/abundance ratios for some rare taxa but lower ratios for most abundant taxa for all the communities, which was further corroborated by the high replication rate (iRep) of most low-abundance population genomes. IMPORTANCE Variation in microbial activity levels is increasingly being recognized as both an important dimension in community function and a complicating factor in sequencing-based survey methods. This study extends previous reports that rare taxa may contribute disproportionately to community activity in some natural environments, showing that this may also hold in artificially maintained model communities with well-described inputs, outputs, and biochemical functions. These results demonstrate that assessment of activity levels using the rRNA/rDNA ratio is robust across taxonomic unit formation methods and is independently corroborated by omics methods. The results also provide insight into the comparative advantages and disadvantages of different taxonomic unit formation methods in amplicon sequencing studies, showing that UNOISE3 provides comparable microbial diversity, structure, and activity information as the 97% sequence similarity method but potentially loses some phylogenetic diversity and creates more "phantom taxa" (which are present in the RNA pool but not the corresponding DNA pool).

Project description:BackgroundIsolated limb perfusion (ILP) is a regional surgical treatment for localized metastatic disease. High doses of chemotherapeutic agents are administered within an extracorporeal circulated isolated extremity, treating the metastasis, while systemic toxicity is avoided. To our knowledge, indexed oxygen supply/demand relationship during ILP has not previously been described. Our aim was to measure and describe oxygen metabolism, specifically oxygen delivery, consumption, and extraction, in an isolated leg/arm during ILP. Also investigate whether invasive oxygenation measurement during ILP correlates and can be used interchangeable with the non-invasive method, near infrared spectroscopy (NIRS).MethodsData from 40 patients scheduled for ILP were included. At six time points blood samples were drawn during the procedure. DO2, VO2, and O2ER were calculated according to standard formulas. NIRS and hemodynamics were recorded every 10 min.ResultsFor all observations, the mean of DO2 was 190±59 ml/min/m2, VO2 was 35±8 ml/min/m2, and O2ER was 21±8%. VO2 was significantly higher in legs compared to arms (38±8 vs. 29±7 ml/min/m2, p=0.02). Repeated measures showed a significant decrease in DO2 in legs (209±65 to 180±66 ml/min/m2, p=<0.01) and in arms (252±72 to 150±57 ml/min/m2, p=<0.01). Significant increase in O2ER in arms was also found (p=0.03). Significant correlation was detected between NIRS and venous extremity oxygen saturation (SveO2) (rrm=0.568, p=<. 001, 95% CI 0.397-0.701). When comparing SveO2 and NIRS using a Bland-Altman analysis, the mean difference (bias) was 8.26±13.03 (p=<. 001) and the limit of agreement was - 17.28-33.09, with an error of 32.5%.ConclusionDO2 above 170 ml/min/m2 during ILP kept O2ER below 30% for all observations. NIRS correlates significant to SveO2; however, the two methods do not agree sufficiently to work interchangeable. Clinical Trial Registration URL: https://www.clinicaltrials.gov. Unique identifier: NCT04460053 and NCT03073304.

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Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

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