Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method

Prest, Emmanuelle I.; Hammes, Frederik; Kötzsch, Stefan; Loosdrecht, M.C.M. van; Vrouwenvelder, Johannes S.

doi:10.1016/j.watres.2013.07.051

Cited by 266 publications

(284 citation statements)

References 40 publications

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“…Therefore, non-culture based methods should be used additionally to support the claim that the animals are germ-free. Recently, flow cytometry has become an established and highly valued technique for the microbial analysis of aquatic samples (Díaz et al 2010, Forberg et al 2011, Prest et al 2013. The greatest advantage of flow cytometry, besides speed, reproducibility and large sample sizes, is that the quantification and identification of organisms that formerly could not be detected by culture techniques is now achievable (Hernlem & Ravva 2007).…”

Section: Discussionmentioning

confidence: 99%

Germ-free sea bass Dicentrarchus labrax larval model: a valuable tool in the study of host-microbe interactions

Schaeck

Swaef²,

Broeck³

et al. 2016

Dis. Aquat. Org.

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A thorough understanding of host−microbe interactions is crucial for more efficient disease management in the marine larviculture industry. As demonstrated in terrestrial animal research, gnotobiotic systems (involving animals cultured in germ-free conditions or inoculated with known microorganisms) are excellent tools to extend our understanding of the mechanisms involved in host−microbe interactions and allow the evaluation of new treatments for diseases. In this study, we introduce a germ-free European sea bass Dicentrarchus labrax larval model, independent of the continuous addition of antimicrobial agents. This model has an experimental set-up that allows addition of live feed to the larvae without compromising the germ-free status. This model will facilitate and render aquaculture research more effective in terms of mitigation fish larval diseases.KEY WORDS: Aquaculture · Gnotobiotic · Pathogen · Larvae Resale or republication not permitted without written consent of the publisherDis Aquat Org 117: [177][178][179][180][181][182][183][184][185] 2016 towards more strict regulations on the use of antibiotics in the aquaculture sector (Suba singhe et al. 2001, Romero et al. 2012, hence calling for alternative, sustainable methods to which the aquaculturist can resort for preventing and controlling disease outbreaks. Several environmentally friendly prophylactic disease treatments have been the focus of recent research, i.e. probiotics, prebiotics, vaccines, immunostimulants or antimicrobial peptides (Merrifield & Ringø 2014).A judicious and scientifically supported application of the abovementioned alternatives warrants a thorough testing of their efficacy and safety under standardized and controlled experimental conditions (Smith et al. 2003). However, the stochastic colonization of larvae by microorganisms may hinder the establishment of a reproducible experimental set-up by generating high inter-individual and inter-batch variability in the composition of the standing microbial community. Hence, from a microbiological point of view, iterating experimental conditions using conventional animals is almost impossible (Fjellheim et al. 2012). The development of test systems in which the researcher has complete control over the microbial community structure, by adopting a germ-free or gnotobiotic model, was revolutionary in this respect. As already demonstrated in multiple terrestrial animal studies, gnotobiotic models are an excellent tool to extend our understanding of (1) the nutritional requirements of host organisms, (2) host−microbe interactions and (3) host metabolic functions (Gordon & Pesti 1971, Wostmann 1996, Marques et al. 2005. Despite the significant increase in the use of fish as experimental animals during the last decades (Marques et al. 2005, Schaeck et al. 2013) and the stressed importance of raising aquatic organisms gnotobiotically (Bates et al. 2006), the current know-how of rearing gnotobiotic aquatic organisms is much more limited compared to the more traditional mammalian l...

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Section: Discussionmentioning

confidence: 99%

Germ-free sea bass Dicentrarchus labrax larval model: a valuable tool in the study of host-microbe interactions

Schaeck

Swaef²,

Broeck³

et al. 2016

Dis. Aquat. Org.

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“…[8]. The results obtained with cytometer were converted to the quantity of microorganisms found in 1 ml of water before the test, taking into account the dilution used.…”

Section: Determination Of the Quantity Of Microorganisms By Flow Cytomentioning

confidence: 99%

“…The fluorescence can be detected in Green (FL1), Orange (FL2) and Red (FL3) [10]. In the microbiology of water, it is preferable to use fluorescent dye (520 nm), the intensity of the fluorescence is directly related to the content of nucleic acids in the cells to be detected, as well as green fluorescence (533 nm) [8]. This type of colours to distinguish organisms with high and low content of nucleic acid that is living, inactive [3].…”

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confidence: 99%

Newmethod for evaluation of the microbiological quality of water

2015

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Determination of the microbiological quality of water is one of the central tasks of environmental engineering. Reliability and accuracy of the results depends largely on the sampling system and the methodology carried signs. Typically, they are used traditional indirect methods, breeding: plate method or test tube fermentation. In the methods, seed stocks, it is assumed that each individual cell of bacteria transferred to a solid substrate colony grows. Thus, in fact, the result of the study determines the number of units capable of forming colonies, which is not equivalent to the amount of the living. Even larger errors are subject to the results of the analysis of test tube fermentation, which can only be the most probable number of bacteria in a given volume. It is therefore only estimating the number of microorganisms. Moreover, indirect methods are extremely time consuming (about 1 -7 days of). Flow cytometry is an analytical technique that allows for the rapid measurement of scattered light and fluorescence signals emitted by cells exposed respectively. Allows for a qualitative and quantitative assessment of the physical and biological properties of the cells in a short time. The article presents the results of research on the amount of bacterial cells in different water: surface, groundwater, rainwater and tap water using flow cytometry and compare this results obtained with traditional methods of reference. The microbial results in all the tested waters by flow cytometry are much higher than the amount of the reported conventional methods.

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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.…”

mentioning

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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Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method

Cited by 266 publications

References 40 publications

Germ-free sea bass Dicentrarchus labrax larval model: a valuable tool in the study of host-microbe interactions

Germ-free sea bass Dicentrarchus labrax larval model: a valuable tool in the study of host-microbe interactions

Newmethod for evaluation of the microbiological quality of water

Absolute quantification of microbial taxon abundances

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