The Maintenance of Microbial Community in Human Fecal Samples by a Self-made Cost-effective Preservation Buffer

doi:10.21203/rs.3.rs-218780/v1

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The Maintenance of Microbial Community in Human Fecal Samples by a Self-made Cost-effective Preservation Buffer

https://doi.org/10.21203/rs.3.rs-218780/v1

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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.

General Microbiology

microbial community

human fecal samples

cost-effective preservation buffer

microbiome filed

Mounting evidence has intensively recognized the pivotal role of gut microbiome in the maintenance of host health and the onset or progression of diseases^1–3, especially for those investigations on human population, which have provoked great inspirations for health management and therapeutics explorations^4,5. In the last decade, the rapid development of high-throughput sequencing techniques and powerful analytical methods make considerable contributions to the flourishment of microbiome filed via interpreting the massive datasets^6–8, thereby providing a comprehensive elucidation to the crosstalk between versatile microbiota and host. As a prerequisite, sequencing samples are of great significance for the subsequent microbiome investigations. Due to the noninvasiveness and readily acquisition, fecal sample is the commonly used as a proxy of gut microbiota, such as 16S ribosomal RNA amplicon sequencing, shotgun metagenomic sequencing and metabolomics. Given that the bacterial community in fresh samples could largely represents the real microbial structure, feces after defecation are recommended for the immediate extraction of microbial DNAs, in order to avoid the contamination of exogenous microorganisms and overgrowth of bacterial, which may substantially alter microbiota consortia, ultimately resulting in a misleading for the following programs.

In practice, freshly collected samples usually go through a freezing step prior to the systematical sequencing and analysis, especially for the large-scale investigations. 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^10–16, ranging from urban cities (e.g. human, laboratory organisms, companion animals) to remote areas (e.g. population cohorts and wildlife), making it infeasible to supply refrigeration. More recently, close attentions have been paid up to the stabilization of sequencing samples, which was overlooked for quite a long time.

At present, couples of preservation methods have been developed to maintain microbiome^17,18, for instance, there are dozens of commercial products available in the market, including OMNIgene GUT kit, Norgen, and RNAlater. Among them, OMNI kit was deemed as a better alternative to the ‘gold standard’ by different teams^17–20, according to the assessment of microbial structure and composition, whereas the total expenditure would be considerable for large-scale studies. Other researchers have acknowledged the beneficial 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 inflammability of ethanol) or a high cost. Besides, the storage temperature and duration are another two important factors to affect the performance of microbial DNA. The former mainly includes ambient temperature and low temperature (including 4°C, -20°C, and − 80°C). Samples are usually kept at either ambient temperature or 4°C for a temporal storage, prior to an ultimate cryopreservation. Storage time distinctly varies from several hours to weeks or months according to project schedules, availability of freezing facilities and logistics into considerations. Thus, it is meaningful to investigate how preservation time or temperature impact sample stabilities, which bear great missions for the subsequent omics-related studies. Overall, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand, especially for population cohorts-based investigations, despite of the presence of commercial products.

In this study, we conducted shotgun metagenomic sequencing to evaluate the effects of storage temperature (including ambient temperature, 4°C, -20°C, and − 80°C) on fecal samples donated by volunteers, in terms of microbial structure and composition. We also verified a self-made preservation buffer (PB) capable of maintaining fecal samples at ambient temperature up to 4 weeks and even enduring an extra high temperature condition which mimics temperature fluctuations in summer logistics. Moreover, PB enabled the stabilization of human saliva samples as well, exhibiting suitability to other sample types. Taken together, PB buffer may be a better choice to stabilize sequencing samples when facing the shortage of freezing facilities or logistics constrains.

Short-term preservation does not alter fecal microbiota community independent of the storage temperature. Since stool samples commonly experience a temporary storage after sampling, we wondered whether a short period storage would significantly affect microbiome stabilization. Nine volunteers donated fecal samples and each sample was divided into four aliquots to be stored for 4h at ambient temperature (AT), 4°C, -20°C and − 80°C, respectively. -80°C preservation was used as the control method. Post temporary storage, microbial DNAs were extracted from each aliquot followed by shotgun sequencing. As showed in Fig. 1A, we found a similar α-diversity among all groups estimated by indices of Shannon, Simpson and Evenness, though values were slightly higher in AT and 4°C groups than freezing groups (-20°C and − 80°C), which was possibly caused by the introduction of experimental microorganisms or the altered growth affected by atmospheric oxygen. These results indicated that microbial structures in stool samples were not significantly affected by storage temperature under a short-term preservation within 4 hours. Principle coordinate analysis (PCoA) further revealed that samples across four different temperature groups tended to overlap each other, exhibiting a high similarity of bacterial structure (Fig. 1B). Analogously, the Euclidean distance of microbial communities to -80°C group was not altered among ambient temperature, 4°C and − 20°C groups (Fig. 1C).

As for bacterial composition, we then analyzed the relative abundances of dominant genera and species. At the genera level, six out of nine samples (including samples from No.1 to 5 and No.8) showed an almost identical compositional profile across four temperature conditions (Fig. 1D), suggesting few perturbations caused by different temperatures to microbial community. Other samples displayed a disordered pattern: No.6 sample had a similar composition under AT, 4°C and − 80°C temperatures, the profiles of No.7 sample at AT, 4°C and − 20°C were different from that at -80°C, while for No.9 sample, ambient temperature storage changed the genera abundance, obviously distinct from other temperatures. Regarding the species profiling, we observed a relatively consistent pattern with the genera abundance (Fig. 1E).

Collectively, our results implied that a temporary storage no longer than 4 hours could stabilize both microbial structure and composition in human fecal samples, independent of the storage temperature. These findings also provided a hint that the temperature factor did not matter for a temporary preservation (e.g. <4 hours), thus a short-period storage may be a reliable practice to be adopted at the absence of refrigerators and liquid nitrogen.

A self-prepared preservation buffer (PB) enables to stabilize fecal microbial consortia. It is widely accepted that storage at room temperature can destroy the microbial consortia in sequencing samples. In accordance, we indeed observed the remarkable changes of microbial structure and composition caused by in donated fecal samples under room temperature, as compared with snap-frozen samples with LN (Fig. 2). Importantly, the alterations apparently occurred after even one-day RT treatment, though the storage period last up to 4 weeks. Miguel and colleagues once reported a nucleic acid preservation (NAP) buffer could maintain the quantity and quality of RNA and DNA from mammal samples under field conditions²³. We then evaluated whether this lab-prepared preservation buffer (abbreviated as PB in the context) could be applied to stabilize microbial communities under ambient temperature (PB-AT) and high temperature (PB-HT) mimicking the temperature fluctuation during summer transportation. For community diversity, contrary to RT storage, PB application marginally decreased the values of Shannon, Simpson and Evenness indices (Fig. 2A), compared with LN group and the reduce tended to be stable after 3-day preservation. Meanwhile, PB-HT group samples which underwent 2-week PB preservation followed by an extra 50°C treatment displayed a highly similar profile of bacterial diversity with PB-AT group (Fig. 2A). These results clearly presented the inhibitory role of PB buffer in microbiota blooming under room temperature.

Based on distance analysis (Fig. 2B), the relative distance of AT group to LN group was approximately twice further than that of PB-applied group (for Bray-Curtis, 0.6 vs 0.3; for Euclidean, 0.4 vs 0.2), and the spearman coefficients of two PB groups were apparently higher than AT samples when relative to LN group (Fig. 2B). Moreover, samples stored at ambient temperature (included in the blue ellipse) drifted heavily from LN-frozen samples (the red dots) (Fig. 2C), while PB-preserved samples (mainly embraced in the pink ellipse) tended towards LN group, exhibiting that PB-treated groups were in close proximity to LN group regarding the microbial structures.

Furthermore, we performed compositional analysis at the genera and species levels and found that one-day AT preservation substantially altered genera abundance in microbial community, distinct from the pattern displayed in LN group (Fig. 3A), indicating samples should avoid storage at room temperature, even merely for one day. Conversely, PB maintained the genus composition pattern to a large extent, with some mild perturbations in the proportion of genera, e.g. the increase in Prevotella, Bacteroides and Eubacterium; the reduction in Megamonas and Megasphaera (Fig. 3A), yet far less than the destruction of composition resulted from ambient temperature storage. We also observed a great similarity in the relative abundance of dominant genera between PB-AT and PB-HT groups (Fig. 3A). Consistently, the species composition exhibited an analogous pattern to the observed genera profile (Fig. 3B).

According to these findings, the utilization of self-made PB buffer could facilitate to stabilize microbiome in human fecal samples, promoting samples eligible for the following complicated analysis.

PB buffer is suitable for human saliva to maintain microbiota community. Aside from we human beings, investigations in the burgeoning microbiome filed 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 mucosa¹, thereby provoking the multifarious samples and the corresponding exploration of preservation methods. In this study, we next tested whether the protective effects of PB would be retained in another sample type. Taking saliva as an example, we collected saliva samples from five volunteers and each sample was divided into 2 aliquots for − 80°C cryopreservation and PB buffer preservation at ambient temperature, respectively. According to the aforementioned results of fecal samples preserved with PB buffer (Fig. 2,3), one-week storage was used as the representative duration in this experiment. Compared with − 80°C condition, treatment with PB buffer did not change the indices of Shannon, Simpson, and Evenness of microbial community, as depicted in Fig. 4A, indicating the α-diversity of saliva microbiota was maintained by PB buffer. PCoA analysis also showed that PB-treated samples clustered together with frozen samples (Fig. 4B). Our results consolidated that application of PB buffer could stabilize the structure of saliva microbiome.

For microbial composition, PB group displayed a similar overall profile of dominant genera with that of control samples (Fig. 4C), although there were some alterations such as the relative abundance of Neisseria and Haemophilus was decreased and Actinomyces was increased post PB storage as compared to each − 80°C control. At the species level, the similar trends could be observed as well (Fig. 4D). Based on the above analysis, we concluded that the self-made PB buffer could prominently maintain the microbial consortia in saliva samples, exhibiting an acceptable suitability.

Researchers have reached a clear consensus regarding the regulatory role of microbiome, particularly for gut microbiota, in human health and diseases based on breakthrough studies in the prevailing field^3–5. Microbiome-related potential therapies and nutritional interventions have already been promoted such as fecal microbiota transplantation (FMT)^25,26 and next-generation probiotics²⁷. Undoubtedly, the powerful meta-omics techniques and the concomitant analytical methods immensely drive the inspiring findings of the intimate crosstalk between gut microbes and human health^1,6,15. 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 well-characterized by various subjects and a vast geographical coverage^10–16, 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. Moreover, we proved a self-made preservation buffer (PB) could not only keep fecal microbiota stable at ambient temperature up to several weeks but also enable samples to endure a high temperature condition mimicking temperature variations in summer logistics. Besides, the PB buffer exhibited suitability for human saliva, thus making it possible to be a valuable option if required.

Preservation methods naturally came out as the storage demand was raised. Researchers usually evaluate the effects of various methods on microbial community via 16S ribosomal RNA sequencing, which is a popular tool to interpret the complex interplay between microbiome and host. This technology provides a comprehensive understanding of the microbial structure, taxonomic composition, yet several limitations apparently exist, for instance, the limited taxonomy resolution down to species level²⁸. Our previous work also confirmed that 16S sequencing provided less consistent data with whole-genome shotgun sequencing (WGSS), as well as the inaccurate functional predication (data not shown). As a result, the mere focus on microbial structure may not thoroughly mirror the stabilization of microorganisms in sequencing samples. Due to the prosperous development of meta-omics, several groups have recently applied meta-proteomics or metabolomics individually or in combination with 16S sequencing to examine the effects of preservation methods on microbiome^20,29. Herein, we performed the more advanced technology, WGSS, instead of 16S rRNA amplicon sequencing, to assess each preservation factor. Besides the measurement of α-diversity parameters, our analysis also contained the relative abundance patterns of dominant genera and species, aiming to provide more information.

Nowadays, two major types of preservation agents are available to store sequencing samples, including commercial items and simply self-prepared buffers. To our knowledge, there are approximately a dozen commercial products with certain reputation such as OMNIgene GUT, Norgen, RNAlater, Shield, MGIEasy, Longsee, fecal occult blood test (FOBT) cards, and fecal immunochemical test (FIT) tubes^17,18,22,30. Among them, OMNIgene GUT kit displays a better performance to stabilize stool samples as multiple teams have reported^17,20. Interestingly, RNAlater did not constantly inhibit the shift in microbial community after sampling, unable to sustain its well-known role in RNA protection^17,18. Two medical tools (FOBT and FIT) were also indicated as acceptable choices for fecal sample collection and storage in future sequencing²². Apart from these, ethanol has been mentioned most in previous studies to evaluate its impacts on microbial profiles in fecal samples^21,22,30. More specifically, 100% ethanol preferably stabilized microbiome structure as compared to 75% ethanol^14,30. Unfortunately, the abroad application of ethanol does not come as the reagent shows up in the list of dangerous goods, thereby leading to the restricted transportation. Other teams once revealed DMSO-related preservation buffers exhibited few interruptions to α- and β-diversity of gut microbiota in the feces of Japanese adults³¹, even performed as efficiently as snap freezing did in coral specimens²⁴. Miguel once demonstrated a self-made nucleic acid preservation (NAP) buffer could protect RNA and DNA from mammal samples under field conditions²³. Subsequently, this buffer was employed by Sebastian et al to prevent the shift of microbial community after sampling in sheep feces³². In our study, we investigated how the self-prepared preservation buffer (PB buffer, namely NAP buffer) affects the gut microbial consortia in human stool and saliva samples, aiming to explore a time-saving and cost-efficient preservative. Our findings indicated PB buffer to be a valuable alternative to the ‘gold standard’ of sample handling, when facing difficulties in suppling freezing equipment and lipid nitrogen. We draw this conclusion basically based on three reasons: (1) PB buffer maintains fecal microbiota up to several weeks at ambient temperature, with no significant shift in community structure, as well as the genera and species composition, as compared to flash freezing samples. (2) PB buffer favors fecal samples to endure several high temperature days, mimicking temperature changes during summer transportation or storage. (3) The buffer is readily available and cost-saving as the used reagents are common chemicals. Still, we need to verify the protective roles of PB buffer across both sample types and species for an abroad promotion, though the human saliva microbiome was not significantly affected by PB buffer treatment. Additionally, a large population is quite necessary to provide solid evidence for the verification work, in light of the obvious individual variances among volunteers. Furthermore, other powerful approaches and analytical methods may facilitate the assessment and discovery of effective preservatives, which could involve in the future research planning.

In summary, we demonstrated that the human fecal microbiota was not perturbed in a temporary storage, no matter which storage temperature was chose, including ambient temperature, 4°C, -20°C, and − 80°C. For a long-term preservation, a lab-prepared preservation buffer (PB) could properly maintain microbial profiles for up to four weeks at room temperature, and even sustain an extra five high temperature days. Besides, PB was also favorable to protect the microbial community in human saliva. Therefore, our work provided a suitable alternative to immediate freezing for the subsequent fecal microbiome analysis, particularly under conditions where refrigeration and cold chain transportation are not feasible.

Sample collection and storage. Freshly collected feces and saliva samples were immediately divided into aliquots according to the experiment design. All aliquots were then performed under each indicated conditions.

(1) For evaluation of storage temperature in a short-term (4h) preservation, nine volunteers donated the fresh stool samples and each sample was divided into 4 parts for the following conditions: storage at ambient temperature (AT), refrigerating at 4°C, immediate freezing at -20°C or -80°C.

(2) For the usage of preservation buffer (PB), fecal samples were contributed by 3 participants and each sample was split into 14 aliquots, belonging to the following groups: the liquid nitrogen (LN)-treated group, the AT group (preserved for 1 day, 3 days, 1 week, 2 and 4 weeks, respectively), the PB-AT groups (underwent with the same duration as AT group), the PB-high temperature (HT) group (including a pre-storage with PB buffer for 2 weeks followed by an extra 50°C preservation for 3/4/5 days, respectively).

(3) For the assessment of suitability, each sample from 5 volunteers was divided into 2 parts for the cryopreservation at -80°C and the application of PB buffer at ambient temperature for one week.

As for the preservative, the recipe of self-made preservation buffer (PB) was consisted of 20 mM ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, 25 mM sodium citrate trisodium salt dihydrate, 5.3M ammonium sulfate, referring to the previous reported NAP buffer with some mild modifications ²³.

DNA extraction. The microbial genomic DNA of human stool samples and saliva samples were extracted using DNeasy PowerSoil kit (Qiagen) according to the manufacturer’s instructions. Fecal samples were washed with sterilized PBS (Solarbio), and centrifuged at 13,000 g for 5 min to separate the supernatant. The pellets were saved for the following DNA extraction. The extracted DNA was then evaluated by 1% agarose gel electrophoresis. DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific) and Qubit 3.0 fluorometer (Thermo Fisher Scientific).

Shotgun sequencing. Library preparation for shotgun sequencing was performed using the KAPA HyperPlus Library Preparation kit (KAPA Biosystems) for fragmentation of input DNA following the manufacturer’s instructions. The libraries were quantified by using KAPA Library Quantification Kits (KAPA Biosystems) following the manufacturer’s instructions. Libraries were constructed with an insert size of approximately 350 bp, followed by high-throughput sequencing to obtain paired-end reads with 150 bp in the forward and afterward directions. Shotgun sequencing was performed on an Illumina NovaSeq 6000 System (Illumina). Cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing and hybridization of the sequencing primers were performed according to the workflow indicated by Illumina.

Data analysis. The alpha and beta diversities were calculated by Mothur1.30.2. The alpha diversity was assessed by the indices of Shannon, Simpson and Evenness. The beta diversity was assessed by Bray-Curtis distances and Eluclidean distances. The principal coordinate analysis (PCoA) was calculated based the OTU table, and the difference between groups was analyzed by Adonis test. The relative abundance of each taxon was calculated by Metaphlan2 software.

Statistical analysis. Data were showed as means ± SEM. GraphPad Prism was used for the statistical analysis. The significance among groups was assessed by one-way ANOVA followed by Newman-Keuls post hoc tests. P < 0.05 was considered statistically significant.

Acknowledgements

We are grateful for participations of volunteers in this study. This work was supported financially by the National Natural Science Foundation of China (81973217), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018RC350014), CAMS Innovation Fund for Medical Sciences (CIFMS) 2016-I2M-3-015.

Author contributions

C.W. and B.Z. conceived the project, designed the experiments. T.C., T.Z. and Y.P. performed experiments. C.W. and W.X. prepared figures and manuscript with the help of Y.Y. and F.Z. H. G. and C.W. analyzed the sequencing data. Q.W. and L.W. assisted in the experiment design and discussion. All authors read and approved the manuscript.

Competing interests

T. C., W. X., T. Z., Y. P., H. G., Q. W., L. W. and B. Z. are employees of Beijing QuantiHealth Technology Co., Ltd. The other authors declare they have no competing interests.

Ethics approval and consent to participate

The volunteers contributing stool and saliva samples were recruited as part of research protocol number 2020LL-3 approved by the Third Affiliated Hospital of Qiqihar Medical University. The study was performed in accordance with the Helsinki Declaration. The informed consent was acquired from all volunteers recruited into the study.

Data availability

The raw data for fecal and mock microbial communities acquired by both 16S and shotgun WMS have been deposited in the NCBI Sequence Read Archive (SRA) database (Accession Numbers: No. XXXXX)

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Competing interest reported. T. C., W. X., T. Z., Y. P., H. G., Q. W., L. W. and B. Z. are employees of Beijing QuantiHealth Technology Co., Ltd. The other authors declare they have no competing interests.

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The Maintenance of Microbial Community in Human Fecal Samples by a Self-made Cost-effective Preservation Buffer

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