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Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8 weeks
... The low cost and global accessibility of ethanol have created much interest in testing the efficacy of ethanol for microbial community stabilization (Cock et al. 2019). However, the results of different studies indicated a poor performance associated with the use of 75% ethanol solution in the preservation of human fecal samples (Hale et al. 2015;Song et al. 2016;Cock et al. 2019). Therefore, we have selected absolute ethanol as one of the preservatives. ...
... There was exceptionally higher DNA yield in absolute ethanol-fixed samples than all the other methods. Conversely, past studies have identified low DNA yield as one potential drawback of using ethanol in fecal samples (Vlčková et al. 2012;Hale et al. 2015). However, Song et al. (2016) showed that absolute ethanol could preserve the DNA of canine and human fecal samples with high reliability in accordance with our observation. ...
... More importantly, Pseudomonadales was the dominant order in all the methods (average relative abundance, 46%), except in RAE preserved samples where Vibrionales were more prevalent (average relative abundance, 42.39%), followed by Rhodobacterales. In consonance with our observation, Hale et al. (2015) and Hickl et al. (2019) hypothesized that different human fecal sample preservation methods could exhibit some biases towards or against certain microbiological groups. Moreover, no such studies were available in the larval microbiome for comparison. ...
Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P < 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at − 20 °C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species.
Key points
• First report on effects of preservation methods on fish larval microbiome profiles.
• First report on activity ranking of preservation methods based on metagenomics metrics.
• Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.
... DNA and RNA deteriorate rapidly after collection when kept at room temperature (Cardona et al., 2012), while the chemistry of existing stabilizing solutions has also demonstrated an impact on the recovery of genomic microbial content, resulting in a source of bias (Wu et al., 2019;Chen et al., 2020). Despite these conflicting results and challenges, a few principles are currently well acknowledged by the scientific community: avoid freeze-thaw cycles and temperature fluctuations throughout the preservation process (Cardona et al., 2012;Gorzelak et al., 2015;Thomas et al., 2015;Kim et al., 2017) ; when possible, shorten the transportation time; and freezing samples at -20°C or -80°C provides an optimal solution when immediate analysis of fresh sample is not an option (Wu et al., 2010;Bahl et al., 2012;Carroll et al., 2012;Fouhy et al., 2015;Gorzelak et al., 2015;Hale et al., 2015;Voigt et al., 2015;Shaw et al., 2016;Sinha et al., 2016;Song et al., 2016;Hickl et al., 2019;Wu et al., 2019). ...
... For example, RNAlater was until recently the most commonly used buffer for metagenomic studies (Nechvatal et al., 2008;Cardona et al., 2012;Dominianni et al., 2014;Choo et al., 2015;Flores et al., 2015;Thomas et al., 2015;Song et al., 2016). However, its suitability for microbiome analysis has been extensively reviewed, as some studies claim that it results in reduced overall DNA yields and reduces the detection/abundance of bacterial taxa (Dominianni et al., 2014;Choo et al., 2015;Gorzelak et al., 2015;Hale et al., 2015;Sinha et al., 2016;Hickl et al., 2019). Our results did not show reduced DNA yield compared to other preserved solutions but did show significant alterations in the recovered microbiota compared to their references, thus agreeing with previous studies that RNAlater is not an optimal preservation method. ...
... Our results did not show reduced DNA yield compared to other preserved solutions but did show significant alterations in the recovered microbiota compared to their references, thus agreeing with previous studies that RNAlater is not an optimal preservation method. We came to the same conclusion for FTA card as Hale et al. (Hale et al., 2015), who demonstrated that FTA card (and RNAlater)preserved samples were the least similar to fresh samples, while in contrast, Sinha et al. (Sinha et al., 2016) recommended the use of FTA card for short-term storage, demonstrating that it provides reproducible, stable, and accurate data across laboratories (over 4-day storage). The longer storage time in our protocol might have contributed to our discordant results. ...
Microbiotas play critical roles in human health, yet in most cases scientists lack standardized and reproducible methods from collection and preservation of samples, as well as the choice of omic analysis, up to the data processing. To date, stool sample preservation remains a source of technological bias in metagenomic sequencing, despite newly developed storage solutions. Here, we conducted a comparative study of 10 storage methods for human stool over a 14-day period of storage at fluctuating temperatures. We first compared the performance of each stabilizer with observed bacterial composition variation within the same specimen. Then, we identified the nature of the observed variations to determine which bacterial populations were more impacted by the stabilizer. We found that DNA stabilizers display various stabilizing efficacies and affect the recovered bacterial profiles thus highlighting that some solutions are more performant in preserving the true gut microbial community. Furthermore, our results showed that the bias associated with the stabilizers can be linked to the phenotypical traits of the bacterial populations present in the studied samples. Although newly developed storage solutions have improved our capacity to stabilize stool microbial content over time, they are nevertheless not devoid of biases hence requiring the implantation of standard operating procedures. Acknowledging the biases and limitations of the implemented method is key to better interpret and support true associated microbiome patterns that will then lead us towards personalized medicine, in which the microbiota profile could constitute a reliable tool for clinical practice.
... For example, clinical or diet studies involving human subjects usually require the subjects to perform the sampling themselves at their homes, where they do not have access to liquid nitrogen (43,44). It can be difficult to maintain sample integrity also in resourcelimited fieldwork conditions (45) or out in a wild animal's environment, where there is no access to liquid nitrogen and cold storage (46). Even in the laboratory, immediate freezing in liquid nitrogen can be difficult. ...
... RNAlater is a popular storage solution that has been shown to be effective at preserving DNA in gut microbiome studies, with negligible differences compared to freezing (50,51). Ethanol (95% or absolute) may also be suitable for the preservation of nucleic acids in microbiome samples before taxonomic profiling as long as it is used consistently (45,46,52). However, there are studies in which these storage solutions significantly biased the downstream results (53), particularly in RNA sequencing studies (54), and thus great care should be taken when selecting a preservation method. ...
... Alcohol preservation is a common method in which biological specimens are preserved by dehydration. Hale et al. (46) found that absolute ethanol worked as well as immediate freezing of DNA for preserving samples prior to metagenomics analysis. The effectiveness of ethanol as a preservation treatment depends on its concentration. ...
A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for approaches that measure gene expression, such as metaproteomics, which is used to identify and quantify proteins in microbiomes. Intestinal microbiome samples are typically stored by flash-freezing and storage at 280°C, but some experimental setups do not allow for immediate freezing of samples. In this study, we evaluated methods to preserve fecal microbiome samples for metaproteomics analyses when flash-freezing is not possible. We collected fecal samples from C57BL/6 mice and stored them for 1 and 4 weeks using the following methods: Flash-freezing in liquid nitrogen, immersion in RNAlater, immersion in 95% ethanol, immersion in a RNAlater-like buffer, and combinations of these methods. After storage, we extracted protein and prepared peptides for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis to identify and quantify peptides and proteins. All samples produced highly similar metaproteomes, except for ethanol-preserved samples that were distinct from all other samples in terms of protein identifications and protein abundance profiles. Flash-freezing and RNAlater (or RNAlater-like treatments) produced metaproteomes that differed only slightly, with less than 0.7% of identified proteins differing in abundance. In contrast, ethanol preservation resulted in an average of 9.5% of the identified proteins differing in abundance between ethanol and the other treatments. Our results suggest that preservation at room temperature in RNAlater or an RNAlater-like solution performs as well as freezing for the preservation of intestinal microbiome samples before metaproteomics analyses.
... One of each of these subsample pairs were immediately frozen (untreated subsample), and the other subsamples were soaked in 99.5% ethanol for 1 day before freezing (ethanol-treated subsample) (Table S5). Treating the feces with ethanol is a method sometimes used in stable isotope, DNA, and hormone analyses to prevent molding and to facilitate the preservation of biomolecules (Frantzen et al. 1998;Hale et al. 2015;Phillips and O'Connell 2016;Roeder et al. 2004;Strier and Ziegler 1997). Ethanol treatment was not applied to feces from the oldest female (Gypsy) because of its sticky and liquid texture. ...
... Chemical or mass spectrometric analyses of the ethanolsoluble fraction of feces would elucidate the chemical process occurring with ethanol treatment. Ethanol treatment is sometimes used to prevent molding and to facilitate the preservation of biomolecules (Frantzen et al. 1998;Hale et al. 2015;Phillips and O'Connell 2016;Roeder et al. 2004;Strier and Ziegler 1997), but isotopic effects of ethanol treatment of fecal samples might vary among different species and diets, as well as different conditions of ethanol treatment, such as the concentration of ethanol, duration of treatment, and relative volume of ethanol to the sample mass. Thus, it is important to investigate the effect of a specific ethanol treatment for feces obtained from the study subject population when comparing ethanol-treated and untreated samples. ...
... Thus, it is important to investigate the effect of a specific ethanol treatment for feces obtained from the study subject population when comparing ethanol-treated and untreated samples. Since fecal samples obtained from wild animals were sometimes stored in ethanol for macroscopic observation of food remains, DNA analysis, or hormone analyses in several previous studies (e.g., Frantzen et al. 1998;Hale et al. 2015;Roeder et al. 2004;Strier and Ziegler 1997), such knowledge can broaden the applicability of stable isotope analysis to the remaining fecal materials in these studies. ...
Estimating stable isotopic offset values is crucial for dietary reconstructions. Although research into stable isotope ecology of wild nonhuman primates is increasing overall, only a minority of studies involve laboratory experiments. This study is the first to report the carbon and nitrogen stable isotopic offset values in hair and feces of orangutans. During an experiment lasting 1 week, the weight of each consumed food item was recorded for each of six captive Bornean orangutan (Pongo pygmaeus) individuals. The food, hair, and fecal samples were collected for a few days, and their stable carbon and nitrogen isotope ratios were measured using an elemental analyzer/isotope ratio mass spectrometer. Subsamples of feces were treated with ethanol during the preservation process. Monte Carlo analyses showed that the 95% confidence intervals (CIs) of the carbon and nitrogen offset values between hair and diet were +0.9‰ to +3.9‰ and +2.3‰ to +4.5‰, respectively. The 95% CIs of the carbon and nitrogen offset values between feces and diet were −3.7‰ to −0.9‰ and +0.3‰ to +2.7‰, respectively. The effect of ethanol treatment on the stable isotope ratios of feces was unclear and inconclusive. The computed offset values of hair in captive orangutans are similar to those reported in other nonhuman primates, although those of feces showed greater interspecies variations. The offset values estimated in this study contribute to isotopic studies into the feeding ecology of free-ranging orangutans who are critically endangered in most wild settings.
... For example, clinical or diet studies involving human subjects usually require the subjects to perform the sampling themselves at their homes where they do not have access to liquid nitrogen [43,44]. It can be difficult to maintain sample integrity also in resource-limited fieldwork conditions [45] or out in a wild animal's environment where there is no access to liquid nitrogen and cold storage [46]. Even in the laboratory, immediate freezing in liquid nitrogen can be difficult. ...
... RNAlater™ is a popular storage solution that has been shown to be effective at preserving DNA in gut microbiome studies, with negligible differences compared to freezing [50,51]. Ethanol (95% or absolute) may also be suitable for the preservation of nucleic acids in microbiome samples before taxonomic profiling as long as it is used consistently [45,46,52]. However, there are studies in which these storage solutions significantly biased the downstream results [53], particularly in RNA sequencing studies [54] and thus great care should be taken when selecting a preservation method. ...
... Hale and colleagues [46] found that absolute ethanol worked as well as immediate freezing of DNA for preserving samples prior to metagenomics analysis. The effectiveness of ethanol as a preservation treatment depends on its concentration. ...
A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for approaches that measure gene expression, such as metaproteomics, which is used to identify and quantify proteins in microbiomes. Intestinal microbiome samples are typically stored by flash freezing and storage at −80°C, but some experimental set-ups do not allow for immediate freezing of samples. In this study, we evaluated methods to preserve fecal microbiome samples for metaproteomics analyses when flash freezing is not possible. We collected fecal samples from C57BL/6 mice and stored them for 1 and 4 weeks using the following methods: flash-freezing in liquid nitrogen, immersion in RNAlater™, immersion in 95% ethanol, immersion in a RNAlater-like buffer, and combinations of these methods. After storage we extracted protein and prepared peptides for LC-MS/MS analysis to identify and quantify peptides and proteins. All samples produced highly similar metaproteomes, except for ethanol-preserved samples that were distinct from all other samples in terms of protein identifications and protein abundance profiles. Flash-freezing and RNAlater™ (or RNAlater-like treatments) produced metaproteomes that differed only slightly, with less than 0.7% of identified proteins differing in abundance. In contrast, ethanol preservation resulted in an average of 9.5% of the identified proteins differing in abundance between ethanol and the other treatments. Our results suggest that preservation at room temperature in RNAlater™, or an RNAlater-like solution, performs as well as freezing for the preservation of intestinal microbiome samples before metaproteomics analyses.
Importance
Metaproteomics is a powerful tool to study the intestinal microbiome. By identifying and quantifying a large number of microbial, dietary, and host proteins in microbiome samples, metaproteomics provides direct evidence of the activities and functions of microbial community members. A critical step for metaproteomic workflows is preserving samples before analysis because protein profiles are susceptible to fast change in response to changes in environmental conditions (air exposure, temperature changes, etc.). This study evaluated the effects of different preservation treatments on the metaproteomes of intestinal microbiome samples.
... Immediate cryopreservation at − 80 °C or snap freezing with liquid nitrogen (LN) is considered as the 'gold standard' of sample preservation 7,9 . However, microbiome studies in the present era have already intensively expanded in terms of geographic region and species diversity [10][11][12][13][14][15][16] , ranging from urban cities (e.g. human, laboratory organisms, companion animals) to remote areas (e.g. ...
... However, the total expenditure on sampling process would be considerable for large-scale studies using OMNI kit (20-25$/kit). Interestingly, other researchers have explored several self-made recipes to evaluate their preservative effects on microbiome, such as ethanol 14 , DMSO-EDTA salt solution (DESS) 21 and 4% paraformaldehyde solution 22 . Hale et al. found that 100% ethanol could preserve fecal microbial composition to a similar extent to fresh samples of spider monkey 14 . ...
... Interestingly, other researchers have explored several self-made recipes to evaluate their preservative effects on microbiome, such as ethanol 14 , DMSO-EDTA salt solution (DESS) 21 and 4% paraformaldehyde solution 22 . Hale et al. found that 100% ethanol could preserve fecal microbial composition to a similar extent to fresh samples of spider monkey 14 . A study involving Japanese adults revealed DESS did not considerably affect the fecal microbiota and OUT profiles 21 . ...
In the burgeoning microbiome field, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. The proper preservation of samples before being processed is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4 h, independent of the storage temperature. We also verified a previous reported self-made preservation buffer (PB buffer) could not only preserve fecal microbiota at room temperature up to 4 weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize samples if neither freezing facilities nor liquid nitrogen is available.
... Other researchers have acknowledged the bene cial effects of several self-made recipes (e.g. 95% ethanol) on the preservation of fecal samples without altering α-diversity of microbial community 14,21,22 . Nonetheless, the widely-spread application of these agents is still impeded due in part to the inconvenience (e.g. the in ammability of ethanol) or a high cost. ...
... PB buffer is suitable for human saliva to maintain microbiota community. Aside from we human beings, investigations in the burgeoning microbiome led have reached to a wide variety of subjects such as model organisms, companion animals, wildlife, and marine organisms 12,14,16,24 . Mammalian microbiota not only vastly colonizes in host gastrointestinal tracts but also resides within or on the body, including lung, oral mucosa, skin and vaginal mucosa 1 , thereby provoking the multifarious samples and the corresponding exploration of preservation methods. ...
... In recent years, accumulating attentions have been paid to a long-neglected fact that samples guarantee the reliability of studies via providing stable microbiome. Given that longitudinal investigations in the current microbiota era are wellcharacterized by various subjects and a vast geographical coverage [10][11][12][13][14][15][16] , the development and utilization of preservation methods are particularly important to hold samples eligible for the consequent sequencing analysis. In the present work, we performed shotgun sequencing and demonstrated that in a short-term storage, microbial consortia in human fecal samples were substantially maintained, independent of the accompanied storage temperature. ...
In the burgeoning microbiome filed, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. Maintaining sequencing samples is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that in a short-term storage, microbial consortia in human fecal samples were substantially maintained, independent of the storage temperature. We also verified a self-made preservation buffer could not only maintain fecal microbiota at ambient temperature up to several weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize sequencing samples if neither freezing facilities nor liquid nitrogen is available.
... Contradictory results are also observed in the use of RNAlater. One study shows that fecal samples preserved with RNAlater closely resemble fresh samples (Horng et al., 2018), whereas the least similarity in microbial composition and abundance with fresh samples are reported in another study (Hale et al., 2015). In addition, significant differences are also observed in microbial profile of identical samples that are processed and sequenced at two research centers (Sinha et al., 2016;Penington et al., 2018). ...
... Commercial stabilizer for preserving fecal samples at room temperatures are usually suggested due to the fact of sampling with no access to immediate freezing at −20 • C or below or freezing transportation, especially in remote areas. Although the efficacy of some commercial stabilizers on maintaining the original status of gut microbiome has been compared, the uncertainty still exists because of the inconsistent observations among some reports (Hale et al., 2015;Sinha et al., 2016). Previous studies suggested that inter-individual variation from donors superseded that introduced by storage conditions (Guo et al., 2016;Bundgaard-Nielsen et al., 2018;Penington et al., 2018), however, the impacts of different storage conditions on microbiome profile are not well characterized. ...
... Although −80 • C or GT reagents stored samples have been used as controls for evaluating the effectiveness of preservation methods in previous reports (Flores et al., 2015;Song et al., 2016;Han et al., 2018), the efficacy of these preservation conditions are still inconclusive because of the inconsistent results (Horng et al., 2018). Thus, we allocated a part of freshly collected fecal samples as the fresh control, in addition to −80 • C. Stabilization of fecal samples with ethanol is an easy and economical way, and therefore different concentrations of ethanol were used for fecal sample preservation in previous reports including 100% ethanol for spider monkey (Hale et al., 2015), 95% ethanol for human or dog (Song et al., 2016), and 70% ethanol for canine fecal sample storage (Horng et al., 2018). However, inconsistent results were reported previously. ...
Gut dysbiosis is heavily involved in the development of various human diseases. There are thousands of publications per year for investigating the role of gut microbiota in diseases. However, emerging evidence has indicated the frequent data inconsistency between different studies, which is largely overlooked. There are many factors that can cause data variation and inconsistency during the process of microbiota study, in particular, sample storage conditions and sequencing process. Here, we systemically evaluated the impacts of six fecal sample storage conditions (three non-commercial storage protocols, −80°C, −80°C with 70% ethanol (ET_−80°C), 4°C with 70% ethanol (ET_4°C), and three commercial storage reagents, OMNIgeneGUT OMR-200 (GT) and MGIEasy (MGIE) at room temperature, and Longsee at 4°C (LS) on gut microbiome profile based on 16S rRNA gene sequencing. In addition, we also investigated the impacts of storage periods (1 and 2 weeks, or 6 months) and sequencing platform on microbiome profile. The efficacy of storage conditions was evaluated by DNA yield and quality, α and β diversity, relative abundance of the dominant and functional bacteria associated with short-chain fatty acid (SCFA) production, and BAs metabolism. Our current study suggested that −80°C was acceptable for fecal sample storage, and the addition of 70% ethanol had some benefits in maintaining the microbial community structure. Meanwhile, we found that samples in ET_4°C and GT reagents were comparable, both of them introduced some biases in α or β diversity, and the relative abundance of functional bacteria. Samples stored in MGIE reagent resulted in the least variation, whereas the most obvious variations were introduced by LS reagents. In addition, our results indicated that variations caused by storage condition were larger than that of storage time and sequencing platform. Collectively, our study provided a multi-dimensional evaluation on the impacts of storage conditions, storage time periods, and sequencing platform on gut microbial profile.
... OPEN ACCESS further complicated when a lack of good road networks to study sites is thrown into the mix. For studies that involve one-time sampling, other storage methods such as the use of 95% or 100% ethanol [76,77] can be adopted. However, such storage methods may not be suitable for longitudinal studies due to possible reduction in DNA yield [77]. ...
... For studies that involve one-time sampling, other storage methods such as the use of 95% or 100% ethanol [76,77] can be adopted. However, such storage methods may not be suitable for longitudinal studies due to possible reduction in DNA yield [77]. ...
The gut microbiome of neonates, infants, and toddlers (NITs) is very dynamic, and only begins to stabilize towards the third year of life. Within this period, exposure to xenobiotics may perturb the gut environment, thereby driving or contributing to microbial dysbiosis, which may negatively impact health into adulthood. Despite exposure of NITs globally, but especially in Africa, to copious amounts and types of xenobiotics – such as mycotoxins, pesticide residues, and heavy metals – little is known about their influence on the early-life microbiome or their effects on acute or long-term health. Within the African context, the influence of fermented foods, herbal mixtures, and the delivery environment on the early-life microbiome are often neglected, despite being potentially important factors that influence the microbiome. Consequently, data on in-depth understanding of the microbiome–exposome interactions is lacking in African cohorts. Collecting and evaluating such data is important because exposome-induced gut dysbiosis could potentially favor disease progression.
... There have been some case studies assessing the effect of various unfrozen preservatives for fecal samples [28][29][30]. At the same time, for insects, conditions are different because insects are compact individuals with chitin exoskeletons which make the samples hard to be mixed with preservative. ...
... In other words, microbial communities preserved by RNAlater might be invalid over 30 days, maybe because such preservative is hard to fully permeate into insects to perform functions effectively. This consequence is similar to those of fecal sample preservation studies in which the effect of RNAlater is often not stable [28][29][30]. However, it will still be recommended when using the insect samples for multiple purposes such as transcriptome sequencing when preserving for 4 weeks or shorter. ...
Bacterial symbionts of insects have been shown to play important roles in host fitness. However, little is known about the bacterial community of Tessaratoma papillosa which is one of the most destructive pests of the well-known fruits Litchi chinensis Sonn and Dimocarpus longan Lour in Oriental Region, especially in South-east Asia and adjacent areas. In this study, we surveyed the bacterial community diversity and dynamics of T. papillosa in all developmental stages with both culture-dependent and culture-independent methods by the third-generation sequencing technology. Five bacterial phyla were identified in seven developmental stages of T. papillosa. Proteobacteria was the dominant phylum and Pantoea was the dominant genus of T. papillosa. The results of alpha and beta diversity analyses showed that egg stage had the most complex bacterial community. Some of different developmental stages showed similarities, which were clustered into three phases: (1) egg stage, (2) early nymph stages (instars 1–3), and (3) late nymph stages (instars 4–5) and adult stage. Functional prediction indicated that the bacterial community played different roles in these three phases. Furthermore, 109 different bacterial strains were isolated and identified from various developmental stages. This study revealed the relationship between the symbiotic bacteria and the development of T. papillosa, and may thus contribute to the biological control techniques of T. papillosa in the future.
... In this study, the specimen collection cards (i.e. GenCollect and GenSaver) stored at room temperatures for 10 weeks showed excellent stability when compared to the immediately frozen cards, although other studies detected lower amounts of DNA among fecal samples from humans and animals collected on FTA cards (Whatman) after several weeks (25,28). Also consistent with previous findings, we found that specimen collection cards tended to differ in bacterial taxa composition and OTUs than when using the putative gold standard (8,22,28), but we did not observed higher Shannon and inverse Simpson. ...
... GenCollect and GenSaver) stored at room temperatures for 10 weeks showed excellent stability when compared to the immediately frozen cards, although other studies detected lower amounts of DNA among fecal samples from humans and animals collected on FTA cards (Whatman) after several weeks (25,28). Also consistent with previous findings, we found that specimen collection cards tended to differ in bacterial taxa composition and OTUs than when using the putative gold standard (8,22,28), but we did not observed higher Shannon and inverse Simpson. Firmicutes, often spore formers, were the most represented phylum in samples collected with specimen collection cards, supporting the hypothesis that chemical cell lysis induced by the card matrix might be one explanation for these differences (22). ...
Background:
Colorectal cancer (CRC) screening programs with fecal sample collection may provide a platform for population-based gut microbiome-disease research. We investigated sample collection and storage methods impact on the accuracy and stability of the V3-V4 region of the 16S rRNA genes and bacterial quantity across seven different collection methods (i.e., no solution, two specimen collection cards, and four types of fecal immunochemical test (FIT) used in four countries) among 19 healthy volunteers.
Methods:
Intraclass correlation coefficients (ICCs) were calculated for the relative abundance of the top three phyla, the most abundant genera, alpha-diversity metrics, and the first principal coordinates of the beta-diversity matrices to estimate the stability of microbial profiles after storage for 7 days at room temperature, 4{degree sign}C, 30{degree sign}C. Additionally, screening for the presence of occult blood in the stool, and accuracy was compared to samples frozen immediately with no solution (i.e. the putative gold standard).
Results:
When compared to the putative gold standard, we observed significant variation for all collection methods. However, inter-individual variability was much higher than the variability introduced by the collection method. Stability ICCs were high ({greater than or equal to}0.75) for FIT tubes that underwent CRC screening procedures. The relative abundance of Actinobacteria (0.65) was an exception and was lower for different FIT tubes stored at 30{degree sign}C (range, 0.41-0.90) and room temperature (range, 0.06-0.94).
Conclusions:
Paper collection cards and different types of FIT are acceptable tools for microbiome measurements.
Impact:
Our findings inform on the utility of commonly used fecal sample collection methods for developing microbiome-focused cohorts nested within screening programs.
... At the same time, alcohols' fixative properties preserve viral and bacterial structures and safely allow further downstream analysis (61)(62)(63). For this reason, ethanol has been commonly used as preserving agent in microbiome samples (64)(65)(66). Due to its low cost and accessibility, ethanol is an attractive option for viral inactivation. However, and to the best of our knowledge, the effect of inactivation using ethanol on microbiome composition and diversity has not been evaluated previously. ...
... The virucidal properties of ethanol are optimal when used at 60% to 80% concentration, as water facilitates protein denaturation (59,70,71). However, previous studies have shown that preserving microbiome samples with 70% ethanol can have detrimental effects on DNA yields, resulting in lower concentrations than freezing or other preservation methods (43,65,66). TRIzol also introduced important changes in the microbiome of different body sites, particularly skin and stool. ...
The global emergence of novel pathogenic viruses presents an important challenge for research, as high biosafety levels are required to process samples. While inactivation of infectious agents facilitates the use of less stringent safety conditions, its effect on other biological entities of interest present in the sample is generally unknown. Here, we analyzed the effect of five inactivation methods (heat, ethanol, formaldehyde, psoralen, and TRIzol) on microbiome composition and diversity in samples collected from four different body sites (gut, nasal, oral, and skin) and compared them against untreated samples from the same tissues. We performed 16S rRNA gene sequencing and estimated abundance and diversity of bacterial taxa present in all samples. Nasal and skin samples were the most affected by inactivation, with ethanol and TRIzol inducing the largest changes in composition, and heat, formaldehyde, TRIzol, and psoralen inducing the largest changes in diversity. Oral and stool microbiomes were more robust to inactivation, with no significant changes in diversity and only moderate changes in composition. Firmicutes was the taxonomic group least affected by inactivation, while Bacteroidetes had a notable enrichment in nasal samples and moderate enrichment in fecal and oral samples. Actinobacteria were more notably depleted in fecal and skin samples, and Proteobacteria exhibited a more variable behavior depending on sample type and inactivation method. Overall, our results demonstrate that inactivation methods can alter the microbiome in a tissue-specific manner and that careful consideration should be given to the choice of method based on the sample type under study.
... There have been some case studies assessing the effect of various unfrozen preservatives for fecal samples [28][29][30]. At the same time, for insects, conditions are different because insects are compact individuals with chitin exoskeletons which make the samples hard to be mixed with preservative. ...
... In other words, microbial communities preserved by RNAlater might be invalid over 30 days, maybe because such preservative is hard to fully permeate into insects to perform functions effectively. This consequence is similar to those of fecal sample preservation studies in which the effect of RNAlater is often not stable [28][29][30]. However, it will still be recommended when using the insect samples for multiple purposes such as transcriptome sequencing when preserving for 4 weeks or shorter. ...
The microbial communities associated with insects play critical roles in many physiological functions such as digestion, nutrition, and defense. Meanwhile, with the development of sequencing technology, more and more studies begin to focus on broader biodiversity of insects and the corresponding mechanisms of insect microbial symbiosis, which need longer time collecting in the field. However, few studies have evaluated the effect of insect microbiome sample preservation approaches especially in different time durations or have assessed whether these approaches are appropriate for both next-generation sequencing (NGS) and third-generation sequencing (TGS) technologies. Here, we used Tessaratoma papillosa (Hemiptera: Tessaratomidae), an important litchi pest, as the model insect and adopted two sequencing technologies to evaluate the effect of four different preservation approaches (cetyltrimethylammonium bromide (CTAB), ethanol, air dried, and RNAlater). We found the samples treated by air dried method, which entomologists adopted for morphological observation and classical taxonomy, would get worse soon. RNAlater as the most expensive approaches for insect microbiome sample preservation did not suit for field works longer than 1 month. We recommended CTAB and ethanol as better preservatives in longer time field work for their effectiveness and low cost. Comparing with the full-length 16S rRNA gene sequenced by TGS, the V4 region of 16S rRNA gene sequenced by NGS has a lower resolution trait and may misestimate the composition of microbial communities. Our results provided recommendations for suitable preservation approaches applied to insect microbiome studies based on two sequencing technologies, which can help researchers properly preserve samples in field works.
... Despite increased interest in characterizing the skin microbiome, efforts to understand how collection and storage methods-factors known to affect gut microbiome samples (Hale et al., 2015;Pollock et al., 2018;Song et al., 2016)-impact samples collected from the skin have been minimal. Methods for skin microbiome sample collection, including swabbing, scraping, or taping the participants' skin or collecting a punch biopsy (Grice et al., 2008;Ogai et al., 2018), are often evaluated independently of storage methods (Benderli et al., 2019;Schowalter et al., 2010), such as freezing or preserving in ethanol or other buffers. ...
... Additionally, because freeze-thaw cycles have been shown to alter and degrade the microbial profile of samples (Cuthbertson et al., 2015) and because freezers are often unavailable or unreliable in field settings, we tested the effectiveness of ethanol as a preservative agent for skin samples. Ethanol is commonly used to preserve fecal samples and is considered to be a low cost, field-friendly method (Hale et al., 2015;Song et al., 2016). If ethanol is also a viable option for skin microbiome samples, it may increase opportunities for skin microbiome research in global field settings. ...
Objectives:
The skin, as well as its microbial communities, serves as the primary interface between the human body and the surrounding environment. In order to implement the skin microbiome into human biology research, there is a need to explore the effects of different sample collection and storage methodologies, including the feasibility of conducting skin microbiome studies in field settings.
Methods:
We collected 99 skin microbiome samples from nine infants living in Veracruz, Mexico using a dual-tipped "dry" swab on the right armpit, palm, and forehead and a "wet" swab (0.15 M NaCl and 0.1% Tween 20) on the same body parts on the left side of the body. One swab from each collection method was stored in 95% ethanol while the other was frozen at -20°C. 16S rRNA amplicon sequencing generated data on bacterial diversity and community composition, which were analyzed using PERMANOVA, linear mixed effects models, and an algorithm-based classifier.
Results:
Treatment (wet_ethanol, wet_freezer, dry_ethanol, and dry_freezer) had an effect (~10% explanatory power) on the bacterial community diversity and composition of skin samples, although body site exhibited a stronger effect (~20% explanatory power). Within treatments, the collection method (wet vs. dry) affected measures of bacterial diversity to a greater degree than did the storage method (ethanol vs. freezer).
Conclusions:
Our study provides novel information on skin microbiome sample collection and storage methods, suggesting that ethanol storage is suitable for research in resource-limited settings. Our results highlight the need for future study design to account for interbody site microbial variation.
... Также предпринимались попытки по хранению биобразцов при комнатной температуре до 24 ч, при этом состав микробиоты, по результатам анализа 16S рРНК, не менялся. На стабильность генома микроорганизмов оказывает влияние активность ДНКазы, что стало поводом для изучения использования различных стабилизаторов: этанол, ЭДТА буферы, а также коммерческие продукты RNAlater, OMNIgene·GUT и FTA cards [29,32]. Однако у них есть определенные недостатки. ...
... Однако у них есть определенные недостатки. Показано, что использование этанола может существенно снизить выход ДНК в сравнении со свежезамороженными образцами [32], применение Трис-ЭДТА буфера и RNAlater может влиять на содержание популяций некоторых родов микробиоты [29]. Использование реагента OMNIgene·GUT позволяло сохранять биообразец до 8 нед. ...
The advances of biomedicine include the new technologies, diagnosis and treatment techniques, as well as the practical use of new types of biological targets, in particular, nucleic acids. Genomic deoxyribonucleic acid (DNA), extracellular DNA (exDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. The use of new technologies for DNA analysis required the development of standardized methods for processing biological samples in order to obtain high-quality DNA samples. The research uses various methods for collecting, preparing samples and storing various DNA-containing biomaterials and isolated DNA, as well as methods for assessing the quality of samples and biobank standards. It is obvious that the use of uniform standards will allow large-scale genetic research on the basis of biobanks and research laboratories. Specialists from professional organizations such as International Society for Biological and Environmental Repositories (ISBER), Biobanking and BioMolecular Resources Research Infrastructure-European Research Infrastructure Consortium (BBMRI-ERIC), European, Middle Eastern & African Society for Biopreservationa and Biobanking (ESBB) and the Russian National Association of Biobanks and Biobanking Professionals.
... Tracheas were aseptically excised, immediately snap frozen in liquid nitrogen at the time of collection, and stored at À80 C and used within 3 mo. Under these storage conditions, microbial profiles in the samples can be preserved for years (11,12). Each frozen trachea was thawed, and the luminal mucosa washed under sterile conditions in a biosafety cabinet by flushing through 2 ml of sterile 13 phosphate-buffered saline (PBS) several times with a pipette. ...
... Except for the nasal wash, the bacterial community compositions captured by tracheal washes and lower respiratory lavage were reproducible, regardless of whether samples were pooled or processed individually (Figs. [12][13][14]. We propose that the nasal cavity was seeded by compositionally different environmental microbiota in each experiment and, therefore, recommend environmental samples to be included in respiratory microbiome sampling. ...
Although poultry microbiome discoveries are increasing due to the potential impact on poultry performance, studies examining the poultry respiratory microbiome are challenging because of the low microbial biomass and uniqueness of the avian respiratory tract, making it difficult to sample enough material for microbial analysis. Invasive sampling techniques requiring euthanasia are currently used to increase microbial mass for the analysis, thus making it impossible to sample individual birds longitudinally. In this study, we compared invasive (nasal wash, upper tracheal wash, lower tracheal wash, and lower respiratory lavage) and noninvasive (tracheal and choanal swabs) respiratory sampling techniques in two independent experiments by using 4-wk-old chickens. We first established the experimental baseline of respiratory microbiota by using invasive techniques to enable reasonable comparisons between sampling methods and between experiments. Although noninvasive sampling (live-bird swabs) resulted in lower 16S ribosomal RNA gene copy numbers compared with invasive sampling, live swabs were able to detect the dominant microbes captured by invasive techniques. Nevertheless, swabs from euthanatized birds were more reflective of the microbiota captured through invasive methods than live swab. Furthermore, from two separate experiments, we also demonstrated that respiratory microbiota sampling is highly reproducible, especially in the trachea and lower respiratory tract. Our study provides new insights and perspectives on decision making when sampling and studying poultry respiratory microbiota.
... Appropriate sample collection and preservation methods are important when investigating symbioses between hosts and their gut microbes [1][2][3][4]. Although the use of entire digestive tracts of freshly obtained specimens is ideal to characterize and capture the full diversity and relative abundances of bacteria residing within the guts [2,[5][6][7], the acquisition of full digestive tracts is often problematic in many vertebrate groups. ...
... The slight reduction in the relative abundance of members of the phyla Bacteroidetes and Actinobacteria in alcohol preserved specimens could be due to degradation of bacterial DNA. In addition, it may be that ethanol adversely affects DNA extraction, but a previous study suggests that this has a minimal impact on microbial community characterizations [4]. The genera that were differentially abundant between alcohol preserved and fresh specimens were inconsistent across different gut regions, suggesting that they are unlikely to be caused by the alcohol preservation, but rather are due to the naturally high variation between individuals in passerine bird gut microbiomes [16,37]. ...
Background Comprehensive studies of wild bird microbiomes are often limited by difficulties of sample acquisition. However, widely used non-invasive cloacal swab methods and under-explored museum specimens preserved in alcohol provide promising avenues to increase our understanding of wild bird microbiomes, provided that they accurately portray natural microbial community compositions. To investigate this assertion, we used 16S rRNA amplicon sequencing of Great tit (Parus major) gut microbiomes to compare 1) microbial communities obtained from dissected digestive tract regions and cloacal swabs, and 2) microbial communities obtained from freshly dissected gut regions and from samples preserved in alcohol for two weeks or two months, respectively. Results We found no significant differences in alpha diversities in communities of different gut regions and cloacal swabs (except in OTU richness between the dissected cloacal region and the cloacal swabs), or between fresh and alcohol preserved samples. However, we did find significant differences in beta diversity and community composition of cloacal swab samples compared to different gut regions. Despite these community-level differences, swab samples qualitatively captured the majority of the bacterial diversity throughout the gut better than any single compartment. Bacterial community compositions of alcohol-preserved specimens did not differ significantly from freshly dissected samples, although some low-abundant taxa were lost in the alcohol preserved specimens. Conclusions Our findings suggest that cloacal swabs, similar to non-invasive fecal sampling, qualitatively depict the gut microbiota composition without having to collect birds to extract the full digestive tract. Secondly, the satisfactory depiction of gut microbial communities in alcohol preserved samples opens up for the possibility of using an enormous resource readily available through museum collections to characterize bird gut microbiomes. The use of extensive museum specimen collections of birds for microbial gut analyses would allow for investigations of temporal patterns of wild bird gut microbiomes, including the potential effects of climate change and anthropogenic impacts. Overall, the utilization of cloacal swabs and museum alcohol specimens can positively impact bird gut microbiome research to help increase our understanding of the role and evolution of wild bird hosts and gut microbial communities.
... Appropriate sample collection and preservation methods are important when investigating symbioses between hosts and their gut microbes [1][2][3][4]. Although the use of entire digestive tracts of freshly obtained specimens is ideal to characterize and capture the full diversity and relative abundances of bacteria residing within the guts [2,[5][6][7], the acquisition of full digestive tracts is often problematic in many vertebrate groups. ...
... The slight reduction in the relative abundance of members of the phyla Bacteroidetes and Actinobacteria in alcohol preserved specimens could be due to degradation of bacterial DNA. In addition, it may be that ethanol adversely affects DNA extraction, but a previous study suggests that this has a minimal impact on microbial community characterizations [4]. The genera that were differentially abundant between alcohol preserved and fresh specimens were inconsistent across different gut regions, suggesting that they are unlikely to be caused by the alcohol preservation, but rather are due to the naturally high variation between individuals in passerine bird gut microbiomes [16,37]. ...
Background: Comprehensive studies of wild bird microbiomes are often limited by difficulties of sample acquisition. However, widely used non-invasive cloacal swab methods and under-explored museum specimens preserved in alcohol provide promising avenues to increase our understanding of wild bird microbiomes, provided that they accurately portray natural microbial community compositions. To investigate this assertion, we used 16S rRNA amplicon sequencing of Great tit (Parus major) gut microbiomes to compare 1) microbial communities obtained from dissected digestive tract regions and cloacal swabs, and 2) microbial communities obtained from freshly dissected gut regions and from samples preserved in alcohol for two weeks or two months, respectively.
Results: We found no significant differences in alpha diversities in communities of different gut regions and cloacal swabs (except in OTU richness between the dissected cloacal region and the cloacal swabs), or between fresh and alcohol preserved samples. However, we did find significant differences in beta diversity and community composition of cloacal swab samples compared to different gut regions. Despite these quantitative differences, swab samples qualitatively captured the bacterial diversity throughout the gut better than any single compartment. Bacterial community compositions of alcohol-preserved specimens did not differ significantly from freshly dissected samples, although some low-abundant taxa were lost in the alcohol preserved specimens.
Conclusions: Our findings suggest that widely used non-invasive cloacal swabs qualitatively depict the gut microbiota composition without having to collect birds to extract the full digestive tract. Secondly, the satisfactory depiction of gut microbial communities in alcohol preserved samples opens up for the possibility of using an enormous resource readily available through museum collections to characterize bird gut microbiomes. The use of extensive museum specimen collections of birds for microbial gut analyses would allow for investigations of temporal patterns of wild bird gut microbiomes, including the potential effects of climate change and anthropogenic impacts. Overall, the utilization of cloacal swabs and museum alcohol specimens can positively impact bird gut microbiome research to help increase our understanding of the role and evolution of wild bird hosts and gut microbial communities.
... Appropriate sample collection and preservation methods are important when investigating symbioses between hosts and their gut microbes [1][2][3][4]. Although the use of entire digestive tracts of freshly obtained specimens is ideal to characterize and capture the full diversity and relative abundances of bacteria residing within guts [2,[5][6][7], the acquisition of full digestive tracts is often problematic in many vertebrate groups. ...
... The slight reduction in the relative abundance of members of the phyla Bacteroidetes and Actinobacteria in alcohol preserved specimens could be due to degradation of bacterial DNA. In addition, it may be that ethanol adversely affects DNA extraction, but a previous study suggests that this has a minimal impact on microbial community characterizations [4]. The genera that were differentially abundant between alcohol preserved and fresh specimens were inconsistent across different gut regions, suggesting that they are unlikely to be caused by the alcohol preservation, but rather are due to the naturally high variation between individuals in passerine bird gut microbiomes [16,37]. ...
Background
Comprehensive studies of wild bird microbiomes are often limited by difficulties of sample acquisition. However, widely used non-invasive cloacal swab methods and under-explored museum specimens preserved in alcohol provide promising avenues to increase our understanding of wild bird microbiomes, provided that they accurately portray natural microbial community compositions. To investigate this assertion, we used 16S rRNA amplicon sequencing of Great tit (Parus major) gut microbiomes to compare 1) microbial communities obtained from dissected digestive tract regions and cloacal swabs, and 2) microbial communities obtained from freshly dissected gut regions and from samples preserved in alcohol for 2 weeks or 2 months, respectively.
Results
We found no significant differences in alpha diversities in communities of different gut regions and cloacal swabs (except in OTU richness between the dissected cloacal region and the cloacal swabs), or between fresh and alcohol preserved samples. However, we did find significant differences in beta diversity and community composition of cloacal swab samples compared to different gut regions. Despite these community-level differences, swab samples qualitatively captured the majority of the bacterial diversity throughout the gut better than any single compartment. Bacterial community compositions of alcohol-preserved specimens did not differ significantly from freshly dissected samples, although some low-abundant taxa were lost in the alcohol preserved specimens.
Conclusions
Our findings suggest that cloacal swabs, similar to non-invasive fecal sampling, qualitatively depict the gut microbiota composition without having to collect birds to extract the full digestive tract. The satisfactory depiction of gut microbial communities in alcohol preserved samples opens up for the possibility of using an enormous resource readily available through museum collections to characterize bird gut microbiomes. The use of extensive museum specimen collections of birds for microbial gut analyses would allow for investigations of temporal patterns of wild bird gut microbiomes, including the potential effects of climate change and anthropogenic impacts. Overall, the utilization of cloacal swabs and museum alcohol specimens can positively impact bird gut microbiome research to help increase our understanding of the role and evolution of wild bird hosts and gut microbial communities.
... Preservation in 70% ethanol was found least effective at stabilizing community structure and yielded similar results to using no preservative measures (Song et al., 2016). Consequently, if ethanol preservation is used, concentrations of at least 95% should be used to reduce preservation biases, particularly where freezing is unavailable and for prolonged sample storage in ambient or sub-optimal conditions (Hale et al., 2015;Song et al., 2016). Additional considerations when selecting between available preservation techniques include potential for conducting further analyses and whether the study is longitudinal in nature. ...
Extensive research in well-studied animal models underscores the importance of commensal gastrointestinal (gut) microbes to animal physiology. Gut microbes have been shown to impact dietary digestion, mediate infection, and even modify behavior and cognition. Given the large physiological and pathophysiological contribution microbes provide their host, it is reasonable to assume that the vertebrate gut microbiome may also impact the fitness, health and ecology of wildlife. In accordance with this expectation, an increasing number of investigations have considered the role of the gut microbiome in wildlife ecology, health, and conservation. To help promote the development of this nascent field, we need to dissolve the technical barriers prohibitive to performing wildlife microbiome research. The present review discusses the 16S rRNA gene microbiome research landscape, clarifying best practices in microbiome data generation and analysis, with particular emphasis on unique situations that arise during wildlife investigations. Special consideration is given to topics relevant for microbiome wildlife research from sample collection to molecular techniques for data generation, to data analysis strategies. Our hope is that this article not only calls for greater integration of microbiome analyses into wildlife ecology and health studies but provides researchers with the technical framework needed to successfully conduct such investigations.
... We preserved fecal material in ethanol because of the lack of other storage possibilities at the field sites. DNA has been successfully isolated and amplified from such fixed samples (Frantzen et al. 1998;Hale et al. 2015), and preserving samples in highly concentrated ethanol at ambient temperatures appears to have little influence on the microbial community (Song et al. 2016). ...
Many studies have demonstrated the importance of symbiotic microbial communities for the host with beneficial effects for nutrition, development, and the immune system. The majority of these studies have focused on bacteria residing in the gastrointestinal tract, while the fungal community has often been neglected. Gut anaerobic fungi of the class Neocallimastigomycetes are a vital part of the intestinal microbiome in many herbivorous animals and their exceptional abilities to degrade indigestible plant material means that they contribute significantly to fermentative processes in the enteric tract. Gorillas rely on a highly fibrous diet and depend on fermentative microorganisms to meet their daily energetic demands. To assess whether Neocallimastigomycetes occur in gorillas we analyzed 12 fecal samples from wild Western lowland gorillas (Gorilla gorilla gorilla) from Dzanga-Sangha Protected Areas, Central African Republic, and subjected potential anaerobic fungi sequences Int J Primatol (2018) 39:567-580 https://doi. to phylogenetic analysis. The clone library contained ITS1 fragments that we related to 45 different fungi clones. Of these, 12 gastrointestinal fungi in gorillas are related to anaerobic fungi and our phylogenetic analyses support their assignment to the class Neocallimastigomycetes. As anaerobic fungi play a pivotal role in plant fiber degradation in the herbivore gut, gorillas might benefit from harboring these particular fungi with regard to their nutritional status. Future studies should investigate whether Neocallimastigomycetes are also found in other nonhuman primates with high fiber intake, which would also benefit from having such highly efficient fermentative microbes.
... Despite the growing evidence that dry preservatives are a suitable means for preserving faecal material [6,8,10,13] their use in microbiome studies has remained limited. Nevertheless, examples of the successful uses of FTA cards for preserving faecal material for gut microbiomes exist for monkeys [14,15], birds [16], and humans [17]. ...
As metagenomic studies continue to increase in size and complexity, they are often required to incorporate data from geographically isolated locations or longitudinal time samples. This represents a technical challenge, given that many of the commonly used methods used for sample collection, storage, and DNA extraction are sensitive to differences related to the time, storage and chemistry involved. FTA cards have been previously proposed as a simple, reliable and cost-efficient method for the preservation of animal faecal microbiomes. In this study, we report a simplified extraction methodology for recovering microbiome DNA from faeces stored on FTA cards and compare its performance to a common alternative means of characterising such microbiomes; namely, immediate freezing of the faeces followed by DNA extraction using the Qiagen PowerSoil DNA isolation kit. Our results show that overall the application of our simplified DNA extraction methodology yields microbial community results that have higher diversity and an expanded core microbiome than that found using the PowerSoil methodology. This suggests that the FTA card extraction method presented here is a viable alternative for metagenomic studies using faecal material when traditional freeze-based storage methods are not feasible.
... Stool samples must be fresh, however, this is usually not possible when remote participants are sampled, thus, self-made preservation buffers or commercial preservation kits can be utilised. Self-made preservation buffers include buffers with preservatives such as ethanol 12,13 , DMSO-EDTA salt solution 14 , EDTA, citrate trisodium salt dihydrate and ammonium sulfate 15 The abundance of critical resistant pathogens, such as extended-spectrum beta-lactamase (ESBL) or carbapenemase-producing Enterobacteriaceae, is often below the detection threshold of direct sequencing. This is a major limitation when characterising the gut resistome 17,18 . ...
In recent years, there has been an increasing interest in the role of the microbiome on human health and disease. Along with this interest, the need for methodological guidance on how to best study the microbiome and its relationship to antibiotic resistance is growing. High-throughput metagenomic sequencing is a powerful tool for investigating the microbiome, and it is becoming increasingly used in clinical research. This review summarizes the existing literature on clinical metagenomics and antimicrobial resistance research and provides guidelines on how to design and conduct studies using this technology. We also discuss some of the challenges and limitations of clinical metagenomics research and suggest future directions for this rapidly evolving field.
... Subsamples for microbiome analysis were fully submerged in anhydrous EtOH for sample stabilization [23][24][25]. Subsamples used for metabolome analysis were treated as previously described. Subsamples were stored at −80 • C until further analysis of the 0 h, 24 h, and 48 h samples. ...
The equine gastrointestinal (GI) microbiota is intimately related to the horse. The objective of the current study was to evaluate the microbiome and metabolome of cecal inoculum maintained in an anaerobic chamber or chemostat batch fermenter, as well as the fecal slurry maintained in an anaerobic chamber over 48 h. Cecal and fecal content were collected from healthy adult horses immediately upon death. Cecal fluid was used to inoculate chemostat vessels (chemostat cecal, n = 11) and vessels containing cecal fluid (anaerobic cecal, n = 15) or 5% fecal slurry (anaerobic fecal, n = 6) were maintained in an anaerobic chamber. Sampling for microbiome and metabolome analysis was performed at vessel establishment (0 h), and after 24 h and 48 h of fermentation. Illumina sequencing was performed, and metabolites were identified via nuclear magnetic resonance (NMR). Alpha and beta diversity indices, as well as individual metabolite concentrations and metabolite regression equations, were analyzed and compared between groups and over time. No differences were evident between alpha or beta diversity in cecal fluid maintained in either an anaerobic chamber or chemostat. The microbiome of the fecal inoculum maintained anaerobically shifted over 48 h and was not comparable to that of the cecal inoculum. Metabolite concentrations were consistently highest in chemostat vessels and lowest in anaerobic fecal vessels. Interestingly, the rate of metabolite change in anaerobic cecal and chemostat cecal vessels was comparable. In conclusion, maintaining an equine cecal inoculum in either an anaerobic chamber or chemostat vessel for 48 h is comparable in terms of the microbiome. However, the microbiome and metabolome of fecal material is not comparable with a cecal inoculum. Future research is required to better understand the factors that influence the level of microbial activity in vitro, particularly when microbiome data identify analogous communities.
... While the number of studies analyzing the host-associated bacterial communities of aquatic invertebrates is increasing, there are few studies analyzing the effects that preservation has on stored specimen's microbiome. Of the few studies previously analyzing the effects that preservation has on any microbiome sample [8,16,17,29], they have primarily focused on preserving fecal specimens of vertebrates rather than preserving the entire host as we did for the aquatic invertebrates sampled in this study. Furthermore, the results of the previous studies were inconclusive as to which storage method would be ideal for microbiome preservation of their samples, leaving the decision to the investigator. ...
Research on the microbiomes of animals has increased substantially within the past decades. More recently, microbial analyses of aquatic invertebrates have become of increased interest. The storage method used while collecting aquatic invertebrates has not been standardized throughout the scientific community, and the effects of common storage methods on the microbial composition of the organism is unknown. Using crayfish and dragonfly nymphs collected from a natural pond and crayfish maintained in an aquarium, the effects of two common storage methods, preserving in 95% ethanol and freezing at −20 °C, on the invertebrate bacterial microbiome was evaluated. We found that the bacterial community was conserved for two sample types (gut and exoskeleton) of field-collected crayfish stored either in ethanol or frozen, as was the gut microbiome of aquarium crayfish. However, there were significant differences between the bacterial communities found on the exoskeleton of aquarium crayfish stored in ethanol compared to those that were frozen. Dragonfly nymphs showed significant differences in gut microbial composition between species, but the microbiome was conserved between storage methods. These results demonstrate that preserving field-collected specimens of aquatic invertebrates in 95% ethanol is likely to be a simple and effective sample preservation method for subsequent gut microbiome analysis but is less reliable for the external microbiome.
... This area has recently gained renewed interest with microbiome and metagenomic studies, showing varying degrees of influence from this step. Consequently, several chemical preservation methods from human and veterinary field studies have been developed and tested for this specific purpose [23][24][25][26][27]. ...
Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives.
This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 10⁶ CFU/mL of both Salmonella and Campylobacter strains stored at −20 °C, 5 °C and 20 °C (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.
... One major advantage of these cards is their ability to remain stable for remarkably long periods of time at room temperature, even for more than 10 years (34). Consistent with other studies, we found that FOBT cards tend to recover a greater diversity of bacterial taxa than other preservation methods (31). Untargeted metabolomics was also used to assess different fecal sample collection methods in this study. ...
Integrative analysis of high-quality metagenomics and metabolomics data from fecal samples provides novel clues for the mechanism underpinning gut microbe-human interactions. However, data regarding the influence of fecal collection methods on both metagenomics and metabolomics are sparse. Six fecal collection methods (the gold standard [GS] [i.e., immediate freezing at 280°C with no solution], 95% ethanol, RNAlater, OMNIgene Gut, fecal occult blood test [FOBT] cards, and Microlution) were used to collect 88 fecal samples from eight healthy volunteers for whole-genome shotgun sequencing (WGSS) and untargeted metabolomic profiling. Metrics assessed included the abundances of predominant phyla and a- and b-diversity at the species, gene, and pathway levels. Intraclass correlation coefficients (ICCs) were calculated for microbes and metabolites to estimate (i) stability (day 4 versus day 0 within each method), (ii) concordance (day 0 for each method versus the GS), and (iii) reliability (day 4 for each method versus the GS). For the top 4 phyla and microbial diversity metrics at the species, gene, and pathway levels, generally high stability and reliability were observed for most methods except for 95% ethanol; similar concordances were seen for different methods. For metabolomics data, 95% ethanol showed the highest stability, concordance, and reliability (median ICCs = 0.71, 0.71, and 0.65, respectively). Taken together, OMNIgene Gut, FOBT cards, RNAlater, and Microlution, but not 95% ethanol, were reliable collection methods for gut metagenomic studies. However, 95% ethanol was the best for preserving fecal metabolite profiles. We recommend using separate collecting methods for gut metagenomic sequencing and fecal metabolomic profiling in large population studies.
... The intestinal bacterial microbiota plays an important role in health and welfare of vertebrates. These bacteria, directly and indirectly, affect the physiology, immune system, and nutrition of the host through different mechanisms of biological coexistence such as mutualism, commensalism, and pathogenicity [1][2][3]. Some benefits provided by the intestinal microbiota are the following: it is a first defense barrier against pathogenic bacteria, promotes intestinal development, enhances fermentative processes to make substrates more easily digested, and produces vitamins for the host [4,5]. ...
The Mexican wolf (Canis lupus baileyi) is an endangered canine. Both Mexico and the United States are currently collaborating to reproduce and reintroduce individuals to their original habitats. However, keeping these wolves in captivity represents a great commitment to meet their basic needs. Diet is a determining factor that is closely related to health and reproductive fitness. The type of diet that is fed to canines in captivity must provide the required nutrients for their development and welfare. The study of the fecal microbiota is a non-invasive way to establish the abundance and diversity of bacterial communities to determine if they are in a healthy condition. We analyzed data from two captive populations of Mexican wolves (i.e., northern and central Mexico) receiving different type of diets (Michilia population: mainly kibble vs. Ocotal population: mainly raw meat). The operational taxonomic units (OTUs) in Michilia resulted in 204 genera and 316 species, while in Ocotal there were 232 genera and 379 species. In the Michilia, dominance of bacteria that degrade carbohydrates was observed (related to kibble diet). In contrast, the Ocotal microbiota was dominated by protein-degrading bacteria (related to raw meat diet). The main outcomes generated in this study should help to enhance the welfare of the captive Mexican wolves to increase its numbers.
... Preservation in OMNIgene.GUT and RNAlater was linked with inflated microbial diversity when compared to frozen samples, and Tris-EDTA usage led to significant differences in the relative abundance of multiple taxa [40]. Additional work on biases introduced due to the use of preservatives has shown that the use of RNAlater leads to significant biases toward the detection of certain taxonomic groups [41,42], and that Stool Nucleic Acid Collection and Preservation tubes (Norgen BioTek Corp.) can result in the over-identification of gram-negative bacteria [43]. Finally, work by Song et al. [37] has shown that the use of FTA cards results in higher levels of taxonomic diversity, but only small consistent differences in taxonomic proportions. ...
Advances in DNA sequencing technology have vastly improved the ability of researchers to explore the microbial inhabitants of the human body. Unfortunately, while these studies have uncovered the importance of these microbial communities to our health, they often do not result in similar findings. One possible reason for the disagreement in these results is due to the multitude of systemic biases that are introduced during sequence-based microbiome studies. These biases begin with sample collection and continue to be introduced throughout the entire experiment leading to an observed community that is significantly altered from the true underlying microbial composition. In this review, we will highlight the various steps in typical sequence-based human microbiome studies where significant bias can be introduced, and we will review the current efforts within the field that aim to reduce the impact of these biases.
-ECi-VLB6JjcxKgnRYA_GNVideo abstract
... However, immediate freezing of faecal samples to inhibit bacterial growth is the optimal procedure for longer term storage. Long term storage of faecal samples at -80°C has been shown to yield microbiota similar to that of fresh samples [106,111,112]. Storage of faecal samples at -20°C also has shown similar efficacy in sample preservation across several studies [105,113,114]. ...
The premature infant gut microbiome plays an important part in infant health and development, and recognition of the implications of microbial dysbiosis in premature infants has prompted significant research into these issues. The approaches to designing investigations into microbial populations are many and varied, each with its own benefits and limitations. The technique used can influence results, contributing to heterogeneity across studies. This review aimed to describe the most common techniques used in researching the preterm infant microbiome, detailing their various limitations. The objective was to provide those entering the field with a broad understanding of available methodologies, so that the likely effects of their use can be factored into literature interpretation and future study design. We found that although many techniques are used for characterising the premature infant microbiome, 16S rRNA short amplicon sequencing is the most common. 16S rRNA short amplicon sequencing has several benefits, including high accuracy, discoverability and high throughput capacity. However, this technique has limitations. Each stage of the protocol offers opportunities for the injection of bias. Bias can contribute to variability between studies using 16S rRNA high throughout sequencing. Thus, we recommend that the interpretation of previous results and future study design be given careful consideration.
... RNAlater could be used as an alternative preservation method, with studies showing no significant difference observed to typically freezing samples at −80 • C, while recovering DNA [47,50], and could therefore be used in addition to freezing at ultra-low temperatures [49]. In contrast, few studies have found opposing outcomes with RNAlater, showing a negative impact on bacterial abundance [51], diversity [52], DNA purity and extraction yields [53,54]. Storage using lyophilization also proved to be ineffective, with failure in preserving species from the Clostridiales order and additionally reducing bacterial diversity [52]. ...
Gastrointestinal (GIT) diseases have risen globally in recent years, and early detection of the host’s gut microbiota, typically through fecal material, has become a crucial component for rapid diagnosis of such diseases. Human fecal material is a complex substance composed of undigested macromolecules and particles, and the processing of such matter is a challenge due to the unstable nature of its products and the complexity of the matrix. The identification of these products can be used as an indication for present and future diseases; however, many researchers focus on one variable or marker looking for specific biomarkers of disease. Therefore, the combination of genomics, transcriptomics, proteomics and metabonomics can give a detailed and complete insight into the gut environment. The proper sample collection, sample preparation and accurate analytical methods play a crucial role in generating precise microbial data and hypotheses in gut microbiome research, as well as multivariate data analysis in determining the gut microbiome functionality in regard to diseases. This review summarizes fecal sample protocols involved in profiling coeliac disease.
... According to the manufacturer, this solution is efficient for preservation for up to 1 day at 37 • C and up to 1 week at 23 • C (room temperature). For samples of monkey feces, Hale et al. (2015) observed that the microbial composition and abundance of samples preserved in RNAlater® differ from the fresh sample. Menke et al. (2017) observed that the NAP solution stabilizes the microbial community better than commercial preservative media (RNAlater® and RNA/DNA Shield™) for samples of sheep feces, making this homemade solution a viable alternative, in terms of both cost and efficiency, for the study of environmental samples from remote areas in the absence of ice or refrigerators. ...
Preservation of environmental samples is an important step in maintaining the original microbial community until the nucleic acid extraction in the laboratory. Here, we collected activated sludge samples to both immediately extract nucleic acids (control) and submit to different storage/preservation methods for 48 h before nucleic acids extraction: room temperature storage, storage on ice, direct storage at −20 °C, rapid freezing in liquid nitrogen followed by storage at −20 °C, and preservation with TRIzol®, RNAlater®, and Nucleic Acid Preservation (NAP) buffers. Bacterial community was compared by high-throughput 16S rRNA gene sequencing from total DNA and RNA. Among the evaluated methods, TRIzol® and rapid freezing with liquid nitrogen were the least indicated, since they led to significant changes in the microbial community structure and abundance of functional groups involved in ammonia metabolism. Compared to control, storage on ice and NAP preserved more than 80% and 75% of genera at the DNA and RNA level respectively, indicating that these methods are the most suitable for storage/preservation of activated sludge samples intended for nucleic acids sequencing.
... Furthermore, several studies have reported that freezing is not always the best practice in the preservation of fecal samples. Choo, Leong, and Rogers (2015) reported that storage at 4°C had similar impacts on fecal microbiome communities, and Hale, Tan, Knight, and Amato (2015) showed that storage in ethanol also had similar impacts. For amplification of mitochondrial and nuclear microsatellite genes, Tende, Hansson, Ottosson, and Bensch (2014) showed that sample storage in ethanol was the best way to obtain a high amplification success rate in most cases, as estimated by their original experiments and review. ...
Fine‐scale estimation of trophic interactions is an important subject in the field of ecology. Diet analysis based on fecal DNA metabarcoding has been accepted as a noninvasive, accurate, and time‐ and cost‐effective tool to determine animal diets. Here, we summarize the trends of fecal metabarcoding studies as well as methodological characteristics using 155 original papers published from 2009 to March 2020. We calculated the frequencies of the methods and conditions used in each experimental procedure and bioinformatics approach. Mammals were the major target taxa for fecal metabarcoding. A few methods or conditions dominated each procedure: sampling, DNA extraction, PCR, sequencing, and bioinformatics, which might be specialized for metabarcoding of degraded fecal DNA. However, the disadvantages of common methods were noted in some studies, and further optimizations are required to obtain more accurate dietary data with high taxonomic resolution and quantitative performance. This review will help fecal metabarcode users, especially new scientists who are considering using fecal metabarcoding in their studies, understand the process and common methods of fecal metabarcoding. We also hope this review will facilitate further technical improvements in this method. We summarize the trends of fecal metabarcoding studies as well as methodological characteristics. Mammals were the major target taxa. A few methods or conditions dominated each procedure, which might be specialized for metabarcoding of degraded fecal DNA. However, further optimizations are required to obtain more accurate dietary data with high taxonomic resolution and quantitative performance.
... There have been other studies that used fecal swabs and standard collection methods in kidney transplantation, [22][23][24] but they used samples that were either collected at home and kept at 4°C rather than using a preservation solution or preserved in RNA later, which has been shown to have decreased DNA purity 25,26 and was poor at maintaining microbiota composition by 16S sequencing. 27 Our pilot study differs in a couple of aspects. ...
The human microbiome encompasses a variety of microorganisms that change dynamically and are in close contact with the body. The microbiome influences health and homeostasis, as well as the immune system, and any significant change in this equilibrium (dysbiosis) triggers both acute and chronic health conditions. Microbiome research has surged, in part, due to advanced sequencing technologies enabling rapid, accurate, and cost-effective identification of the microbiome. A major prerequisite for stool sample collection to study the gut microbiome in longitudinal prospective studies requires standardized protocols that can be easily replicated. However, there are still significant bottlenecks to stool specimen collection that contribute to low patient retention rates in microbiome studies. These barriers are further exacerbated in solid organ transplant recipients where diarrhea is estimated to occur in up to half the patient population. We sought to test two relatively easy sample collection methods (fecal swab and wipes) and compare them to the more cumbersome "gold" standard collection method (scoop) using two different sequencing technologies (16S ribosomal RNA sequencing and shotgun metagenomics). Our comparison of the collection methods shows that both the swabs and the wipes are comparable to the scoop method in terms of bacterial abundance and diversity. The swabs, however, were closer in representation to the scoop and were easier to collect and process compared to the wipes. Potential contamination of the swab and the wipe samples by abundant skin commensals was low in our analysis. Comparison of the two sequencing technologies showed that they were complementary, and that 16S sequencing provided enough coverage to detect and differentiate between bacterial species identified in the collected samples. Our pilot study demonstrates that alternative collection methods for stool sampling are a viable option in clinical applications, such as organ transplant studies. The use of these methods may result in better patient retention recruitment rates in serial microbiome studies.
... Storage time associates with small but signi cant differences in microbiota composition Due to challenges associated with collecting fecal samples in Guinea-Bissau, including unstable electricity supply and limited infrastructure to secure adequate storage at freezing temperatures, fecal samples were collected on FOBT paper and stored at ambient temperature for a longer time period. Existing studies of this procedure has reported a general good performance of the FOBT papers [30][31][32][33][34]. We have recently demonstrated that the storage method used in this study provide microbiota results that are very similar to conventionally stored samples kept at -80°C [28]. ...
Background: Intestinal parasitic infections, caused by helminths and protozoa, are globally distributed and major causes of worldwide morbidity. The gut microbiota may modulate parasite virulence and host response upon infection. The complex interplay between parasites and the gut microbiota is poorly understood, partly due to sampling difficulties in remote areas with high parasite prevalence and burden.
Results: In a large study of children in Bissau, Guinea-Bissau, we found high prevalence of intestinal parasites, including hookworms, Entamoeba spp. and Giardia lamblia. By high-quality sequencing of the 16S rRNA gene of fecal samples stored on filter paper from a total of 1,204 children, we demonstrate that the bacterial microbiota is more or less unaltered by helminth infections, whereas it is shaped by the presence of both pathogenic and non-pathogenic protozoa, including Entamoeba spp. and Giardia lamblia. Within-sample (alpha) diversity remains largely unaffected, whereas overall community composition (beta diversity) is significantly affected by infection with both nonpathogenic Entamoeba coli (R²=0.0131, P= 0.0001) and Endolimax nana (R²=0.00902, P= 0.0001), and by pathogenic Entamoeba histolytica (R²=0.0164, P= 0.0001) and Giardia lamblia (R²=0.00676, P= 0.0001). Heavy infection load with multiple parasite species induces more pronounced shifts in microbiota community than mild ones. A total of 31 bacterial genera across all four major gut bacterial phyla associates with protozoan infection, including increased abundance of Prevotella, Campylobacter and two Clostridium clades (IV and WIVb), and decreased abundance of Collinsella (associated with irritable bowel syndrome), Lactobacillus, Ruminococcus, Veillonella and one Clostridium clade (XVIII). Further, we demonstrate that filter papers are usable for storage of samples at room temperature in large-scale studies where immediate freezing is not possible with minor alterations of microbiota composition.
Conclusion: In the present largest-to-date study, we demonstrate that the fecal bacterial microbiota is shaped by intestinal parasitic infection, with most pronounced associations for protozoan species. Our results provide insights into the interplay between the microbiota and intestinal parasites, which are useful to understand infection outcome and design further studies aimed at optimizing treatment strategies.
... Here, we assessed the influences of several sample preservation conditions commonly used in fecal intestinal microbial profiling as a basis for the design of future sampling methods. In previous studies, researchers used samples stored at -80°C as controls, and even with multiple sample stock solutions and long-term storage, the sequencing results of the microbial communities from the samples showed no significant differences (Fouhy et al., 2015;Hale et al., 2015). Total microbial communities of intestinal microorganisms is greatly influenced by the daily diets of the individual, but they can be affected by environmental factors when the feces are kept at room temperature (David et al., 2014;Hale et al., 2016;Thursby and Juge, 2017); temperature, humidity, and gas can all alter microbial communities. ...
The gut microbiome, which is symbiotic within the human body, assists in human digestion. It plays significant roles in identifying intestinal disease as well as in maintaining a healthy body with functional immune and metabolic activities. To confirm the consistency of fecal intestinal microbial research, it is necessary to study the changes in intestinal microbial flora according to the fecal collection solution and storage period. We collected fecal samples from three healthy Korean adults. To examine the efficacy of fecal collection solution, we used NBgene-Gut, OMNIgene-Gut, 70% ethanol (Ethanol-70%), and RNAlater. The samples were stored for up to two months at room temperature using three different methods, and we observed changes in microbial communities over time. We analyzed clusters of changes in the microbial flora by observing fecal stock solutions and metagenome sequencing performed over time. In particular, we confirmed the profiling of alpha and beta diversity and microbial classification according to the differences in intestinal environment among individuals. We also confirmed that the microbial profile remained stable for two months and that the microbial profile did not change significantly over time. In addition, our results suggest the possibility of verifying microbial profiling even for long-term storage of a single sample. In conclusion, collecting fecal samples using a stock solution rather than freezing feces seems to be relatively reproducible and stable for GUT metagenome analysis. Therefore, stock solution tubes in intestinal microbial research can be used without problems.
... Diagnostic and surveillance testing that require sequence analysis of the sample are more prone to failure given that quality (i.e., stabilization of the genetic materials, requirement for large segments of intact genome) in the sample diminishes compared to testing by conventional PCR and real-time PCR, which are more tolerant of shorter nucleic acid fragments in the test sample. 115 Multiple reports have demonstrated effective use of samples collected onto FTA cards for sequence analysis, including genome sequencing of opossums, rodents, and bats 9 ; microbiome sequencing from feces of spider monkeys (Ateles geoffroyi) 42 and dogs 102 ; genotyping of rotaviruses 109 ; and automated high-throughput genotyping of cattle using whole blood and nasal samples. 69 ...
Evaluation of the diagnostic sensitivity (DSe) and specificity (DSp) of tests for infectious diseases in wild animals is challenging, and some of the limitations may affect compliance with the OIE-recommended test validation pathway. We conducted a methodologic review of test validation studies for OIE-listed diseases in wild mammals published between 2008 and 2017 and focused on study design, statistical analysis, and reporting of results. Most published papers addressed Mycobacterium bovis infection in one or more wildlife species. Our review revealed limitations or missing information about sampled animals, identification criteria for positive and negative samples (case definition), representativeness of source and target populations, and species in the study, as well as information identifying animals sampled for calculations of DSe and DSp as naturally infected captive, free-ranging, or experimentally challenged animals. The deficiencies may have reflected omissions in reporting rather than design flaws, although lack of random sampling might have induced bias in estimates of DSe and DSp. We used case studies of validation of tests for hemorrhagic diseases in deer and white-nose syndrome in hibernating bats to demonstrate approaches for validation when new pathogen serotypes or genotypes are detected and diagnostic algorithms are changed, and how purposes of tests evolve together with the evolution of the pathogen after identification. We describe potential benefits of experimental challenge studies for obtaining DSe and DSp estimates, methods to maintain sample integrity, and Bayesian latent class models for statistical analysis. We make recommendations for improvements in future studies of detection test accuracy in wild mammals.
... ,Knight, & Amato, 2015;Kawada, Naito, Andoh, Ozeki, & Inoue, 2019). The advantages and disadvantages of two common stabilization media, Tris-EDTA, and ethanol-based solutions were previously discussed. ...
Fecal samples collected for microbiome analyses are typically frozen to avoid postcollection changes in microbial composition. eNAT is a guanidine thiocyanate‐based medium that stabilizes microbial DNA and allows safe specimen handling and shipping by inactivating microorganisms. We collected fecal samples (n = 50) from children undergoing hematopoietic stem cell transplantation. We divided samples into three aliquots: (a) stored in RNAlater and immediately transferred to −80°C; (b) stored in eNAT medium and immediately transferred to −80°C; and (c) stored in eNAT medium at ambient temperature (~20°C) for 30 days prior to transfer to −80°C. Mean (standard deviation) Shannon diversity and Chao1 indices in sample aliquots were 2.05 (0.62) and 23.8 (16.6), respectively. Comparing samples frozen immediately in RNAlater to samples frozen immediately in eNAT, there were no differences in Shannon diversity (p = .51), Chao1 richness (p = .66), and overall microbiome composition (p = .99). Comparing eNAT samples frozen immediately to samples stored at ambient temperature, we identified no differences in Shannon diversity (p = .65), Chao1 richness (p = .87), and overall microbiome composition (p = .99). Storage of fecal samples in eNAT at ambient temperature for 30 days did not alter microbiome richness, diversity, or composition. eNAT may be a useful medium for fecal microbiome studies, particularly when cold chain storage is unavailable.
... Over longer time periods, samples can be optimally stored by freezing at -20 °C or -80 °C . Alternatively, they can be freeze-dried or stored in pure ethanol (Hale et al., 2015) or commercially available preservation solutions. ...
The microbes that reside within the equine hindgut create a complex and dynamic ecosystem. The equine hindgut microbiota is intimately associated with health and, as such, represents an area which can be beneficially modified. Synbiotics, supplements that combine probiotic microorganisms with prebiotic ingredients, are a potential means of influencing the hindgut microbiota to promote health and prevent disease. The objective of the current study was to evaluate the influence of an equine probiotic/prebiotic supplement on characteristics of the microbiota and metabolite production in vitro. Equine cecal fluid and fecal material were collected from an abattoir in QC, CAN. Five hundred ml of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, n=11) with either 0g (control) or 0.44g of supplement added at 12h intervals. One hundred ml of cecal fluid (anaerobic cecal, n=15) or 5% fecal slurry (anaerobic fecal, n=6) were maintained in an anaerobic chamber with either 0g (control) or 0.356g of supplement added at the time of vessel establishment. Samples were taken from vessels at vessel establishment (0h), 24h, or 48h of incubation. Illumina sequencing of the V4 region of the 16S rRNA gene and bioinformatics were performed for microbiome analysis. Metabolite data was obtained via NMR spectroscopy. All statistical analyses were run in SAS 9.4. There was no effect of treatment at 24h or 48h on alpha or beta diversity indices and limited taxonomic differences were noted. Acetate, propionate, and butyrate were higher in treated compared to untreated vessels in all methods. A consistent effect of supplementation on the metabolic profile with no discernable impact on the microbiota of these in vitro systems indicates inoculum microbe viability and a utilization of the provided fermentable substrate within the systems. Although no changes within the microbiome were apparent, the consistent changes in metabolites indicates a potential prebiotic effect of the added supplement and merits further exploration.
HIV-infected people develop reproducible disruptions in their gastrointestinal microbiota. Despite the suppression ofHIV viremia via long-term antiretroviral therapy (ART), alterations still occur in gut microbial diversity and the commensal microbiota. Mounting evidence suggests these microbial changes lead to the development of gut dysbiosis—persistent inflammation that damages the gut mucosa—and correlate with various immune defects. In this study, we examined how earlyART intervention influences microbial diversity in SIV-infected rhesus macaques. Using 16S rRNA sequencing, we defined the fecal microbiome in macaques given daily ART beginning on either 3 or 7 d after SIV infection (dpi) and characterized changes in composition, α diversity, and β diversity from before infection through 112 dpi. The dominant phyla in the fecal samples before infection were Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria. After SIV infection and ART, the relative abundance of Firmicutes and Bacteroidetes did not change significantly. Significant reductions in α diversity occurred across time when ART was initiated at 3 dpi but not at 7 dpi. Principal coordinate analysis of samples revealed adivergence in β diversity in both treatment groups after SIV infection, with significant differences depending on the timingof ART administration. These results indicate that although administration of ART at 3 or 7 dpi did not substantially alterfecal microbial composition, the timing of early ART measurably altered phylogenetic diversity.
Sex, age, diet, stress and social environment have all been shown to influence the gut microbiota. In several mammals, including humans, increased stress is related to decreasing gut microbial diversity and may differentially impact specific taxa. Recent evidence from gorillas shows faecal glucocorticoid metabolite concentration (FGMC) did not significantly explain gut microbial diversity, but it was significantly associated with the abundance of the family Anaerolineaceae. These patterns have yet to be examined in other primates, like bonobos ( Pan paniscus ). We compared FGMC to 16S rRNA amplicons for 202 bonobo faecal samples collected across 5 months to evaluate the impact of stress, measured with FGMC, on the gut microbiota. Alpha diversity measures (Chao’s and Shannon’s indexes) were not significantly related to FGMC. FGMC explained 0.80 % of the variation in beta diversity for Jensen–Shannon and 1.2% for weighted UniFrac but was not significant for unweighted UniFrac. We found that genus SHD-231 , a member of the family Anaerolinaceae had a significant positive relationship with FGMC. These results suggest that bonobos are relatively similar to gorillas in alpha diversity and family Anaerolinaceae responses to FGMC, but different from gorillas in beta diversity. Members of the family Anaerolinaceae may be differentially affected by FGMC across great apes. FGMC appears to be context dependent and may be species-specific for alpha and beta diversity but this study provides an example of consistent change in two African apes. Thus, the relationship between physiological stress and the gut microbiome may be difficult to predict, even among closely related species.
The gut microbiota is viewed as the “second genome” of animals, sharing intricate relationships with their respective hosts. Because the gut microbial community and its diversity are affected by many intrinsic and extrinsic factors, studying intestinal microbes has become an important research topic. However, publications are dominated by studies on domestic or captive birds, while research on the composition and response mechanism of environmental changes in the gut microbiota of wild birds remains scarce. Therefore, it is important to understand the co-evolution of host and intestinal bacteria under natural conditions to elucidate the diversity, maintenance mechanisms, and functions of gut microbes in wild birds. Here, the existing knowledge of gut microbiota in captive and wild birds is summarized, along with previous studies on the composition and function, research methods employed, and factors influencing the avian gut microbial communities. Furthermore, research hotspots and directions were also discussed to identify the dynamics of the avian gut microbiota, aiming to contribute to studies of avian microbiology in the future.
In recent years, the number of metagenomic studies increased significantly. Wide range of factors, including the tremendous community complexity and variability, is contributing to the challenge in reliable microbiome community profiling. Many approaches have been proposed to overcome these problems making hardly possible to compare results of different studies. The significant differences between procedures used in metagenomic research are reflected in a variation of the obtained results. This calls for the need for standardisation of the procedure, to reduce the confounding factors originating from DNA isolation, sequencing and bioinformatics analyses in order to ensure that the differences in microbiome composition are of a true biological origin. Although the best practices for metagenomics studies have been the topic of several publications and the main aim of the International Human Microbiome Standard (IHMS) project, standardisation of the procedure for generating and analysing metagenomic data is still far from being achieved. To highlight the difficulties in the standardisation of metagenomics methods, we thoroughly examined each step of the analysis of the human gut microbiome. We tested the DNA isolation procedure, preparation of NGS libraries for next-generation sequencing, and bioinformatics analysis, aimed at identifying microbial taxa. We showed that the homogenisation time is the leading factor impacting sample diversity, with the recommendation for a shorter homogenisation time (10 min). Ten minutes of homogenisation allows for better reflection of the bacteria gram-positive/gram-negative ratio, and the obtained results are the least heterogenous in terms of beta-diversity of samples microbial composition. Besides increasing the homogenisation time, we observed further potential impact of the library preparation kit on the gut microbiome profiling. Moreover, our analysis revealed that the choice of the library preparation kit influences the reproducibility of the results, which is an important factor that has to be taken into account in every experiment. In this study, a tagmentation-based kit allowed for obtaining the most reproducible results. We also considered the choice of the computational tool for determining the composition of intestinal microbiota, with Kraken2/Bracken pipeline outperforming MetaPhlAn2 in our in silico experiments. The design of an experiment and a detailed establishment of an experimental protocol may have a serious impact on determining the taxonomic profile of the intestinal microbiome community. Results of our experiment can be helpful for a wide range of studies that aim to better understand the role of the gut microbiome, as well as for clinical purposes.
Background
The transport and storage of samples in temperatures of minus 80 °C is commonly considered as the gold standard for microbiome studies. However, studies conducting sample collection at remote sites without a reliable cold-chain would benefit from a sample preservation method that allows transport and storage at ambient temperature.
Methods
In this study we compare alpha diversity and 16S microbiome composition of 20 fecal sample replicates from Damaraland mole-rats ( Fukomys damarensis ) preserved in a minus 80 °C freezer and transported on dry ice to freeze-dried samples that were stored and transported in ambient temperature until DNA extraction.
Results
We found strong correlations between relative abundances of Amplicon Sequence Variants (ASVs) between preservation treatments of the sample, no differences in alpha diversity measures between the two preservation treatments and minor effects of the preservation treatment on beta diversity measures. Our results show that freeze-drying samples can be a useful method for cost-effective transportation and storage of microbiome samples that yields quantitatively almost indistinguishable results in 16S microbiome analyses as those stored in minus 80 °C.
The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research.
Objective
Intestinal flora and metabolites are associated with multiple systemic diseases. Current approaches for acquiring information regarding microbiota/metabolites have limitations. We aimed to develop a precise magnetically controlled sampling capsule endoscope (MSCE) for the convenient, non-invasive and accurate acquisition of digestive bioinformation for disease diagnosis and evaluation.
Design
The MSCE and surgery were both used for sampling both jejunal and ileal GI content in the control and antibiotic-induced diarrhoea groups. The GI content was then used for microbiome profiling and metabolomics profiling.
Results
Compared with surgery, our data showed that the MSCE precisely acquired data regarding the intestinal flora and metabolites, which was effectively differentiated in different intestinal regions and disease models. Using MSCE, we detected a dramatic decrease in the abundance of Bacteroidetes, Patescibacteria and Actinobacteria and hippuric acid levels, as well as an increase in the abundance of Escherichia–Shigella and the 2-pyrrolidinone levels were detected in the antibiotic-induced diarrhoea model by MSCE. MSCE-mediated sampling revealed specific gut microbiota/metabolites including Enterococcus , Lachnospiraceae , acetyl-L-carnitine and succinic acid, which are related to metabolic diseases, cancers and nervous system disorders. Additionally, the MSCE exhibited good sealing characteristics with no contamination after sampling.
Conclusions
We present a newly developed MSCE that can non-invasively and accurately acquire intestinal bioinformation via direct visualization under magnetic control, which may further aid in disease prevention, diagnosis, prognosis and treatment.
Predicting how populations and communities of organisms will respond to anthropogenic change is of paramount concern in ecology today. For communities of microorganisms, however, these predictions remain challenging, primarily due to data limitations. Information about long‐term dynamics of host‐associated microbial communities, in particular, are lacking. In this study, we use well‐preserved and freshly collected samples of soft tissue from a marine bivalve host, Donax gouldii, at a single site to quantify the diversity and composition of its microbiome over a decadal timescale. Site‐level measurements of temperature, salinity and chlorophyll a allowed us to test how the microbiome of this species responded to two natural experiments – a seasonal increase in temperature and a phytoplankton bloom. Our results show that ethanol‐preserved tissue can provide high‐resolution information about temporal trends in compositions of host‐associated microbial communities. Specifically, we found that the richness of amplicon sequence variants (ASVs) associated with D. gouldii did not change significantly over time despite increases in water temperature (+1.6°C due to seasonal change) and chlorophyll a concentration (more than nine‐fold). The phylogenetic composition of the communities, on the other hand, varied significantly between all collection years, with only six ASVs persisting over our sampling period. Overall, these results suggest that the diversity of microbial taxa associated with D. gouldii has remained stable over time and in response to seasonal environmental change over the course of more than a decade, but such stability is underlain by substantial turnover in the composition of the microbiome.
Background: Microbiotas play critical roles in human health, yet in most cases scientists lack standardized and reproducible methods. To date, stool sample preservation remains a source of technological bias in metagenomic sequencing, despite newly developed storage solutions. Studying those biases will help in identifying the appropriate stool preservation approach to promote the implementation of standard operating procedure and improve comparability across multiple microbiome studies.
Results: Here, we conducted a comparative study of 10 storage methods for human stool over a 15-day period of storage at fluctuating temperatures. We first compared the performance of each stabilizer with observed bacterial composition variation within the same specimen. Then, we identified the nature of the observed variations to determine which bacterial populations were more impacted by the stabilizer. We found that DNA stabilizers display various stabilizing efficacies and affect the recovered bacterial profiles. Furthermore, our results showed that the bias associated with the stabilizers can be linked to the phenotypical characteristics of the bacterial populations present in the studied samples.
Conclusions: Although newly developed storage solutions have improved our capacity to stabilize stool microbial content over time, they are nevertheless not devoid of biases. We therefore recommend considering the nature of these technological biases before claiming whether some microbes are beneficial or even deleterious to human health.
Many microbial agents have been implicated as contributors to cancer genesis and development, and the search to identify and characterize new cancer-related organisms is ongoing. Modern developments in methodologies, especially culture-independent approaches, have accelerated and driven this research. Recent work has shed light on the multifaceted role that the community of organisms in and on the human body plays in cancer onset, development, detection, treatment, and outcome. Much remains to be discovered, however, as methodological variation and functional testing of statistical correlations need to be addressed for the field to advance.
Here we review all published articles and book chapters, as well as unpublished theses and data of
Ateles geoffroyi diet to (1) summarize the literature; (2) synthesize general feeding patterns; (3)
document plant taxonomic similarity in diet across study sites; and (4) suggest directions for future
research and conservation priorities. We found 22 samples from five countries: Mexico, Guatemala, El
Salvador, Costa Rica and Panama. Tropical wet forest is the most studied habitat (N513 samples),
followed by tropical dry forest (6) and tropical moist forest (3). Most samples have been carried out in
large protected forests. In spite of showing an overall high dietetic diversity (364 species, 76 families),
A. geoffroyi concentrated the majority of feeding time on a few species in the families Moraceae and
Fabaceae. At all study sites fruits were the most common food item in the diet followed by leaves.
Furthermore, a greater variety of food items and less fruit were consumed in forest fragments. These
findings suggest that fruit shortage in fragments results in primates using foods of presumably lower
energetic content such as leaves. Similarity in diet was higher among groups geographically closer to
each other than among distant groups, showing that the floristic and phenological characteristics of the
forest can influence diet composition. We conclude that several years of data are required to fully
describe the dietary list of A. geoffroyi at any one site, as studies of the same group over different years
shared as little as 56% of species. As most populations of A. geoffroyi live in highly fragmented
landscapes, it is crucial to carry out studies in these areas to evaluate (1) changes in diet and activity
patterns that may negatively affect survival; and (2) habitat attributes that may favor their persistence
in altered landscapes.
The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.
Mammalian immune system development depends on instruction from resident commensal micro-organisms. Diseases associated with abnormal immune responses toward environmental and self antigens have been rapidly increasing over the last fifty years. These diseases include inflammatory bowel disease (IBD), multiple sclerosis (MS), type I diabetes (T1D), allergies and asthma. The observation that people with immune mediated diseases house a different microbial community when compared to healthy individuals suggests that pathogenesis arises from improper training of the immune system by the microbiota. However, with hundreds of different microorganisms on our bodies it is hard to know which of these contribute to health and more importantly how? Microbiologists studying pathogenic organisms have long adhered to Koch's postulates to directly relate a certain disease to a specific microbe, raising the question of whether this might be true of commensal-host relationships as well. Emerging evidence supports that rather than one or two dominant organisms inducing host health, that the composition of the entire community of microbial residents influences a balanced immune response. Thus, perturbations to the structure of complex commensal communities (referred to as dysbiosis) can lead to deficient education of the host immune system and subsequent development of immune mediated diseases. Here we will overview the literature that describes the causes of dysbiosis and the mechanisms evolved by the host to prevent these changes to community structure. Building off these studies, we will categorize the different types of dysbiosis and define how collections of microorganisms can influence the host response. This research has broad implications for future therapies that go beyond the introduction of a single organism to induce health. We propose that identifying mechanisms to re-establish a healthy complex microbiota after dysbiosis has occurred, a process we will refer to as rebiosis, will be fundamental to treating complex immune diseases.
Effective means are needed to efficiently collect fecal samples for microbiome analysis in large-scale epidemiological studies. Using twenty-four fecal aliquots prepared from three healthy individuals, we compared the following four fecal sample collection methods for assessment of human gut microbiome: 1) fecal occult blood test cards, held at room temperature for three days, 2) Eppendorf tubes, at room temperature for three days, 3) Eppendorf tubes with RNAlater, at room temperature, and 4) as controls, samples immediately frozen at -80[degree sign]C. The 24 samples were assayed by 16S rRNA gene sequencing to compare overall microbiome structure and taxon distributions according to collection method.
Storing fecal occult blood test card samples at room temperature for three days did not affect total DNA purity and relative 16S rRNA bacterial gene contents, compared with fresh frozen collection. Overall microbiome structure, based on phylogenetic UniFrac index, differed significantly by subject (p = 0.001), but microbiome structure (p = 0.497) and relative abundance of major microbial taxa (phyla) (p > 0.05) did not differ significantly by collection method.
Our findings suggest that low-cost fecal occult blood test card collection may be a feasible means of sample collection for fecal microbiome assessment in large-scale population-based studies.
Mammals have diversified into many dietary niches. Specialized myrmecophagous (ant- and termite-eating) placental mammals represent a textbook example of evolutionary convergence driven by extreme diet specialization. Armadillos, anteaters, aardvarks, pangolins and aardwolves thus provide a model system for understanding the potential role of gut microbiota in the convergent adaptation to myrmecophagy. Here, we expand upon previous mammalian gut microbiome studies by using high-throughput barcoded Illumina sequencing of the 16S rRNA gene to characterize the composition of gut microbiota in 15 species representing all placental myrmecophagous lineages and their close relatives from zoo- and field-collected samples. We confirm that both diet and phylogeny drive the evolution of mammalian gut microbiota, with cases of convergence in global composition, but also examples of phylogenetic inertia. Our results reveal specialized placental myrmecophages as a spectacular case of large-scale convergence in gut microbiome composition. Indeed, neighbour-net networks and beta-diversity plots based on UniFrac distances show significant clustering of myrmecophagous species (anteaters, aardvarks and aardwolves), even though they belong to phylogenetically distant lineages representing different orders. The aardwolf, which diverged from carnivorous hyenas only in the last 10 million years, experienced a convergent shift in the composition of its gut microbiome to become more similar to other myrmecophages. These results confirm diet adaptation to be a major driving factor of convergence in gut microbiome composition over evolutionary timescales. This study sets the scene for future metagenomic studies aiming at evaluating potential convergence in functional gene content in the microbiomes of specialized mammalian myrmecophages.
The gastrointestinal (GI) microbiome contributes significantly to host nutrition and health. However, relationships involving GI microbes, their hosts and host macrohabitats remain to be established. Here, we define clear patterns of variation in the GI microbiomes of six groups of Mexican black howler monkeys (Alouatta pigra) occupying a gradation of habitats including a continuous evergreen rainforest, an evergreen rainforest fragment, a continuous semi-deciduous forest and captivity. High throughput microbial 16S ribosomal RNA gene sequencing indicated that diversity, richness and composition of howler GI microbiomes varied with host habitat in relation to diet. Howlers occupying suboptimal habitats consumed less diverse diets and correspondingly had less diverse gut microbiomes. Quantitative real-time PCR also revealed a reduction in the number of genes related to butyrate production and hydrogen metabolism in the microbiomes of howlers occupying suboptimal habitats, which may impact host health.
Planococcus halocryophilus strain Or1, isolated from high Arctic permafrost, grows and divides at -15 °C, the lowest temperature demonstrated to date, and is metabolically active at -25 °C in frozen permafrost microcosms. To understand how P. halocryophilus Or1 remains active under the subzero and osmotically dynamic conditions that characterize its native permafrost habitat, we investigated the genome, cell physiology and transcriptomes of growth at -15 °C and 18% NaCl compared with optimal (25 °C) temperatures. Subzero growth coincides with unusual cell envelope features of encrustations surrounding cells, while the cytoplasmic membrane is significantly remodeled favouring a higher ratio of saturated to branched fatty acids. Analyses of the 3.4 Mbp genome revealed that a suite of cold and osmotic-specific adaptive mechanisms are present as well as an amino acid distribution favouring increased flexibility of proteins. Genomic redundancy within 17% of the genome could enable P. halocryophilus Or1 to exploit isozyme exchange to maintain growth under stress, including multiple copies of osmolyte uptake genes (Opu and Pro genes). Isozyme exchange was observed between the transcriptome data sets, with selective upregulation of multi-copy genes involved in cell division, fatty acid synthesis, solute binding, oxidative stress response and transcriptional regulation. The combination of protein flexibility, resource efficiency, genomic plasticity and synergistic adaptation likely compensate against osmotic and cold stresses. These results suggest that non-spore forming P. halocryophilus Or1 is specifically suited for active growth in its Arctic permafrost habitat (ambient temp. ∼-16 °C), indicating that such cryoenvironments harbor a more active microbial ecosystem than previously thought.
The gut microbiota of mammals underpins the metabolic capacity and health of the host. Our understanding of what influences the composition of this community has been limited primarily to evidence from captive and terrestrial mammals. Therefore, the gut microbiota of southern elephant seals, Mirounga leonina, and leopard seals, Hydrurga leptonyx, inhabiting Antarctica were compared with captive leopard seals. Each seal exhibited a gut microbiota dominated by four phyla: Firmicutes (41.5 ± 4.0%), Fusobacteria (25.6 ± 3.9%), Proteobacteria (17.0 ± 3.2%) and Bacteroidetes (14.1 ± 1.7%). Species, age, sex and captivity were strong drivers of the composition of the gut microbiota, which can be attributed to differences in diet, gut length and physiology and social interactions. Differences in particular prey items consumed by seal species could contribute to the observed differences in the gut microbiota. The longer gut of the southern elephant seal provides a habitat reduced in available oxygen and more suitable to members of the phyla Bacteroidetes compared with other hosts. Among wild seals, 16 'core' bacterial community members were present in the gut of at least 50% of individuals. As identified between southern elephant seal mother-pup pairs, 'core' members are passed on via vertical transmission from a young age and persist through to adulthood. Our study suggests that these hosts have co-evolved with their gut microbiota and core members may provide some benefit to the host, such as developing the immune system. Further evidence of their strong evolutionary history is provided with the presence of 18 shared 'core' members in the gut microbiota of related seals living in the Arctic. The influence of diet and other factors, particularly in captivity, influences the composition of the community considerably. This study suggests that the gut microbiota has co-evolved with wild mammals as is evident in the shared presence of 'core' members.
DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.Keywords: illumine; barcoded sequencing; QIIME
The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.
Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.
Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.
Animal genomic DNA extracts of sufficient quality to address questions about population biology or behavioural ecology can
be obtained from faeces when adequate extraction procedures are used. The presence of PCR inhibitors in extract products appears
generally the main factor limiting DNA amplification success. We compared DNA amplification success from faeces of a tropical
primate (western lowland gorilla, Gorilla g. gorilla) and a Mediterranean primate (Barbary macaque, Macaca sylvanus) between a standardized extraction technique widely used in animals (QIAamp® stool kit), a technique mainly used in plant species (CTAB) and a new protocol (2CTAB/PCI). Amplification success varied
from 51% to 97%, the highest success being reached with the 2CTAB/PCI protocol in both species.
The large numbers of microorganisms that inhabit mammalian body surfaces have a highly coevolved relationship with the immune
system. Although many of these microbes carry out functions that are critical for host physiology, they nevertheless pose
the threat of breach with ensuing pathologies. The mammalian immune system plays an essential role in maintaining homeostasis
with resident microbial communities, thus ensuring that the mutualistic nature of the host-microbial relationship is maintained.
At the same time, resident bacteria profoundly shape mammalian immunity. Here, we review advances in our understanding of
the interactions between resident microbes and the immune system and the implications of these findings for human health.
DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.
In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.
Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
Reference phylogenies are crucial for providing a taxonomic framework for interpretation of marker gene and metagenomic surveys, which continue to reveal novel species at a remarkable rate. Greengenes is a dedicated full-length 16S rRNA gene database that provides users with a curated taxonomy based on de novo tree inference. We developed a 'taxonomy to tree' approach for transferring group names from an existing taxonomy to a tree topology, and used it to apply the Greengenes, National Center for Biotechnology Information (NCBI) and cyanoDB (Cyanobacteria only) taxonomies to a de novo tree comprising 408,315 sequences. We also incorporated explicit rank information provided by the NCBI taxonomy to group names (by prefixing rank designations) for better user orientation and classification consistency. The resulting merged taxonomy improved the classification of 75% of the sequences by one or more ranks relative to the original NCBI taxonomy with the most pronounced improvements occurring in under-classified environmental sequences. We also assessed candidate phyla (divisions) currently defined by NCBI and present recommendations for consolidation of 34 redundantly named groups. All intermediate results from the pipeline, which includes tree inference, jackknifing and transfer of a donor taxonomy to a recipient tree (tax2tree) are available for download. The improved Greengenes taxonomy should provide important infrastructure for a wide range of megasequencing projects studying ecosystems on scales ranging from our own bodies (the Human Microbiome Project) to the entire planet (the Earth Microbiome Project). The implementation of the software can be obtained from http://sourceforge.net/projects/tax2tree/.