Routine bacterial analysis with automated flow cytometry

Nevel, Sam Van; Koetzsch, Stefan; Weilenmann, Hans Ulrich; Boon, Nico; Hammes, Frederik

doi:10.1016/j.mimet.2013.05.007

Cited by 129 publications

(107 citation statements)

References 11 publications

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“…Next, one colony of each test species was transferred into 9 ml broth and incubated under static conditions overnight at its optimal growth temperature. Subsequently, cultures were transferred (10 % v/v) into fresh broth and allowed to grow for 20 h. Cultures were then diluted to 10 4 intact cells ml À1 in fresh medium, as measured by flow cytometry (BD Accuri C6; Becton, Dickinson and Company) according to Van Nevel et al (2013), before the start of the experiment.…”

Section: Methodsmentioning

confidence: 99%

“…In each well of a 96-well plate, 200 µl of a diluted bacterial suspension (10 4 cells ml À1 ) (see 'Growth assays') were treated with different concentrations of 5-FU (0.1-50 µM) for 24 h at the optimal growth temperature of each test species. After incubation, the number of intact and damaged cells was measured by flow cytometry as described by Van Nevel et al (2013). For this, the samples were diluted in a filter sterile phosphate-buffered solution to obtain cell numbers within the detection range (10 4 -10 6 cells ml À1 ).…”

Section: Methodsmentioning

confidence: 99%

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5-Fluorouracil sensitivity varies among oral micro-organisms

Vanlancker

Vanhoecke

Smet

et al. 2016

Journal of Medical Microbiology

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5-Fluorouracil (5-FU), a commonly used chemotherapeutic agent, often causes oral mucositis, an inflammation and ulceration of the oral mucosa. Micro-organisms in the oral cavity are thought to play an important role in the aggravation and severity of mucositis, but the mechanisms behind this remain unclear. Although 5-FU has been shown to elicit antibacterial effects at high concentrations (>100 µM), its antibacterial effect at physiologically relevant concentrations in the oral cavity is unknown. This study reports the effect of different concentrations of 5-FU (range 0.1-50 µM) on the growth and viability of bacterial monocultures that are present in the oral cavity and the possible role in the activity of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU resistance. Our data showed a differential sensitivity among the tested oral species towards physiological concentrations of 5-FU. Klebsiella oxytoca, Streptococcus salivarius, Streptococcus mitis, Streptococcus oralis, Pseudomonas aeruginosa and Lactobacillus salivarius appeared to be highly resistant to all tested concentrations. In contrast, Lactobacillus oris, Lactobacillus plantarum, Streptococcus pyogenes, Fusobacterium nucleatum and Neisseria mucosa showed a significant reduction in growth and viability starting from very low concentrations (0.2-3.1 µM). We can also provide evidence that DPD is not involved in the 5-FU resistance of the selected species. The observed variability in response to physiological 5-FU concentrations may explain why certain microbiota lead to a community dysbiosis and/or an overgrowth of certain resistant micro-organisms in the oral cavity following cancer treatment.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

5-Fluorouracil sensitivity varies among oral micro-organisms

Vanlancker

Vanhoecke

Smet

et al. 2016

Journal of Medical Microbiology

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“…To our knowledge, there are no descriptive studies that assess the extent to which relative abundances deliver a skewed image of the actual microbial community dynamics. In this study, we combined robust cell density measurements from flow cytometry (Prest et al, 2013;Van Nevel et al, 2013) with the relative abundances derived from 16S rRNA gene amplicon sequencing. We performed two extensive longitudinal surveys on the central water reservoir of a cooling water system.…”

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confidence: 99%

Absolute quantification of microbial taxon abundances

et al. 2016

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High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys. The ISME Journal (2017) 11, 584-587; doi:10.1038/ismej.2016 published online 9 September 2016 Recent advancements in high-throughput sequencing of marker genes, such as the 16S rRNA gene, have provided microbial ecologists the tools to accurately infer the relative composition of microbial communities (Franzosa et al., 2015). This resulted in a widespread application of the technology in longitudinal studies where shifts in community structure are related to environmental variables and functional outputs (Faust et al., 2015;Wilhelm et al., 2015). An inherent limitation of the sequencing technology is that the calculated taxon abundances comprise relative values (Widder et al., 2016). Hence, caution must be taken with the biological interpretation of these values, since inter-sample differences in cell density are not considered. To our knowledge, there are no descriptive studies that assess the extent to which relative abundances deliver a skewed image of the actual microbial community dynamics. In this study, we combined robust cell density measurements from flow cytometry (Prest et al., 2013;Van Nevel et al., 2013) with the relative abundances derived from 16S rRNA gene amplicon sequencing. We performed two extensive longitudinal surveys on the central water reservoir of a cooling water system. This engineered freshwater ecosystem was subjected to highly controlled operational phases (Supplementary Information and data set). We quantified the absolute taxon abundances and assessed whether additional insights could be attained with the combined approach.Based on the sample-specified total cell density, the absolute taxon abundances were calculated for each time point. Individual taxon densities ranged from 0.5 to 1 679 cells per μl. Several inter-taxon differences became apparent by performing ordinary least squares regression analysis between the relative and absolute abundances. We focused on the three most abundant taxa, which ...

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“…Bacterial cell count was performed using a BD Accuri C6 flow cytometer (BD Biosciences, Belgium) according to the live/dead staining protocol described by Van Nevel et al (21). For the analysis, each culture sample contains a 500-l mixture of bacterial culture (5 or 50 l, depending on the dilution factor), fluorescent dyes (5 l; the dye composition is described in reference 22), and sterile physiological solution (0.9% [vol/vol] NaCl).…”

Section: Methodsmentioning

confidence: 99%

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

Ganendra

Muynck

et al. 2014

Appl Environ Microbiol

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e Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such as ammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ؎ 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ؎ 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ؎ 0.03 g of CaCO 3 g of Ca(CHOOH) 2 ؊1 calcium carbonate precipitate yield was obtained when a culture of 10 9 cells ml ؊1 and 5 g of calcium formate liter ؊1 were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry. Microbially induced carbonate precipitation (MICP) is a wellknown process and has been extensively described in the past (1-3). In short, MICP produces carbonate minerals, e.g., calcium carbonate, as a result of alterations in environmental conditions. In nature, examples of MICP include calcite formation in soils (4), limestone caves (5), seas (6), and soda lakes (7). Four different key parameters that govern microbially induced calcium carbonate precipitation are the: (i) concentration of nonprecipitated calcium, (ii) concentration of the total inorganic carbon, (iii) pH, and (iv) availability of nucleation sites for calcium carbonate crystal formation (3). Among the four parameters, bacterial activities mainly influence the pH of the environment (1).MICP is the basis for several biotechnological applications in the construction sector (for a review, see reference 1 and references therein). These include the use of calcium carbonate precipitate to protect concrete surface against the ingress of deleterious substances (e.g., chloride ions) (8) or to heal cracks in aging concrete (9, 10). Among the bacterial activities that can induce calcium carbonate precipitation, urea degradation by heterotrophic bacteria is typically used for applications on building materials. In biogenic urea degradation, urea is transformed to ammonia and carbonate ions to initiate precipitation (11). Bacillus spp. (e.g., B. sphaericus) is the most commonly applied urea degrader for MICP in the construction sector due to several advantages such as the high initial urea degradation rate by the strain and a highly negative potential ...

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Routine bacterial analysis with automated flow cytometry

Cited by 129 publications

References 11 publications

5-Fluorouracil sensitivity varies among oral micro-organisms

5-Fluorouracil sensitivity varies among oral micro-organisms

Absolute quantification of microbial taxon abundances

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

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