Low Bacterial Biomass Research Articles

Tissue-resident bacteria in metabolic diseases: emerging evidence and challenges.

Although the impact of the gut microbiome on health and disease is well established, there is controversy regarding the presence of microorganisms such as bacteria and their products in organs and tissues. However, recent contamination-aware findings of tissue-resident microbial signatures provide accumulating evidence in support of bacterial translocation in cardiometabolic disease. The latter provides a distinct paradigm for the link between microbial colonizers of mucosal surfaces and host metabolism. In this Perspective, we re-evaluate the concept of tissue-resident bacteria including their role in metabolic low-grade tissue and systemic inflammation. We examine the limitations and challenges associated with studying low bacterial biomass samples and propose experimental and analytical strategies to overcome these issues. Our Perspective aims to encourage further investigation of the mechanisms linking tissue-resident bacteria to host metabolism and their potentially actionable health implications for prevention and treatment.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

16S rRNA Next-Generation Sequencing May Not Be Useful for Examining Suspected Cases of Spontaneous Bacterial Peritonitis.

Background and Objectives: Ascites, often associated with liver cirrhosis, poses diagnostic challenges, particularly in detecting bacterial infections. Traditional methods have limitations, prompting the exploration of advanced techniques such as 16S rDNA next-generation sequencing (NGS) for improved diagnostics in such low-biomass fluids. The aim of this study was to investigate whether the NGS method enhances detection sensitivity compared to a conventional ascites culture. Additionally, we aimed to explore the presence of a microbiome in the abdominal cavity and determine whether it has a sterile condition. Materials and Methods: Ten patients with clinically suspected spontaneous bacterial peritonitis (SBP) were included in this study. A traditional ascites culture was performed, and all ascites samples were subjected to 16S ribosomal RNA gene amplification and sequencing. 16S rRNA gene sequencing results were interpreted by comparing them to positive and negative controls for each sample. Results: Differential centrifugation was applied to all ascites samples, resulting in very small or no bacterial pellets being harvested. The examination of the 16S amplicon sequencing libraries indicated that the target amplicon products were either minimally visible or exhibited lower intensity than their corresponding negative controls. Contaminants present in the reagents were also identified in the ascites samples. Sequence analysis of the 16S rRNA gene of all samples showed microbial compositions that were akin to those found in the negative controls, without any bacteria isolated that were unique to the samples. Conclusions: The peritoneal cavity and ascites exhibit low bacterial biomass even in the presence of SBP, resulting in a very low positivity rate in 16S rRNA gene sequencing. Hence, the 16S RNA sequencing method does little to enhance the rate of positive samples compared to traditional culture methods, including in SBP cases.

Nearly (?) sterile avian egg in a passerine bird.

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs.

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon-based sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome.

P-092 Interaction of male and female genital microbiota in infertile couples undergoing assisted reproductive technology

Abstract Study question Is there a crosstalk between the male and female genital microbiota of couples undergoing assisted reproductive technology (ART) and the outcome of this treatment. Summary answer Despite the presence of bacteria linked with bacterial vaginosis in male samples, there appears to be very limited impact on the microbiota of female partners. What is known already In recent years, thanks to advancements in sequencing technologies, the microbiota has been explored extensively and many of its roles in human physiology were deciphered. Compared to its female counterpart, male genital microbiota has been understudied. Presence of bacteria in sperm was generally associated with a pathological condition, but recent reports indicate that the male genital tract is colonized by bacteria and even good quality semen generally contains bacteria. Previously three main semen microbiota profiles were identified, two of these were composed of bacteria, usually found in the vagina, indicating that bacteria could be shared between partners during sexual intercourse. Study design, size, duration Prospective observational study including 65 couples receiving ART between 2018 and 2020 at a single fertility center in Switzerland, to investigate the interaction of male and female reproductive tract microbiota. Samples were collected by swabbing penile skin and seminal fluid as well as vagina and follicular fluid. Participants/materials, setting, methods Samples of 64 infertile couples undergoing ART were collected by direct sampling (vagina and penis glans) or by immersion in the biological fluid (follicular fluid and semen). Follicular fluid was obtained during oocyte pick-up. Sampling of vagina and penis glans (self-sampling) as well as immersion of semen was performed on the same day. 16S rRNA profiling (next generation sequencing) was used to characterize bacterial populations in the samples using a previously developed pipeline. Main results and the role of chance Lactobacillus spp. were highly prevalent in follicular fluid samples and especially in vaginal samples. Generally, penis glans samples were highly colonized by different bacteria and showed the highest alpha diversity. Follicular fluid and semen samples had generally low bacterial biomass. Lactobacillus iners was the most prevalent species in female samples. This is also the case for male samples, although the abundance was lower. For male patients, comparison of beta diversities showed that intra-individual samples were more similar compared to same sample types from different individuals. The opposite was observed for vaginal samples, where inter-individual differences were less marked compared to samples of the same woman. Similar analyses were performed to evaluate relatedness of samples from the same couple, allowing us to estimate the possible impact of microbiota interaction between partners. Despite generally high values, in several cases intra-couple dissimilarities were significantly lower when compared to same sample type of different individuals. Analysis of differently abundant taxa showed, as expected, that Lactobacillus genus was enriched in female samples, along with Atopobium spp. A higher number of genera was specifically enriched in male samples, including Prevotella, Campylobacter and Mobiluncus genera among others. Limitations, reasons for caution Limitations of this study are, that it is only observational and the number of patients included is relatively small. Wider implications of the findings Our results suggest a very limited impact of male microbiota on the female bacterial colonization, despite male samples were enriched with bacterial genera negatively associated with reproduction. Future analysis of the influence of sexual activity on the microbial composition should comprise samples obtained before and after sexual intercourse. Trial registration number not applicable

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding.

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.

Higher off-target amplicon detection rate in MiSeq v3 compared to v2 reagent kits in the context of 16S-rRNA-sequencing

One of the most widely used techniques in microbiota research is 16S-rRNA-sequencing. Several laboratory processes have been shown to impact sequencing results, especially in low biomass samples. Low biomass samples are prone to off-target amplification, where instead of bacterial DNA, host DNA is erroneously amplified. Knowledge on the laboratory processes influencing off-target amplification and detection is however scarce. We here expand on previous findings by demonstrating that off-target amplification is not limited to invasive biopsy samples, but is also an issue in low bacterial biomass respiratory (mucosal) samples, especially when below 0.3 pg/μL. We show that off-target amplification can partly be mitigated by using gel-based library purification methods. Importantly, we report a higher off-target amplicon detection rate when using MiSeq reagent kit v3 compared to v2 (mean 13.3% vs 0.1% off-target reads/sample, respectively), possibly as a result of differences in reagents or sequencing recipes. However, since after bioinformatic removal of off-target reads, MiSeq reagent kit v3 still results in a twofold higher number of reads when compared to v2, v3 is still preferred over v2. Together, these results add to the growing knowledge base on off-target amplification and detection, allowing researchers to anticipate this problem in 16S-rRNA-based microbiome studies involving low biomass samples.

Metagenomic analysis of the microbiome of the upper reproductive tract: combating ovarian cancer through predictive, preventive, and personalized medicine

PurposeWe investigated whether ovarian cancer could alter the genital microbiota in a specific way with clinical values. Furthermore, we proposed how such changes could be envisioned in a paradigm of predictive, preventive, and personalized medicine (PPPM).MethodsThe samples were collected using cotton swabs from the cervical, uterine cavity, fallopian tubes, and ovaries of patients subjected to the surgical procedures for the malignant/benign lesions. All samples were then analyzed by metagenomic shotgun sequencing. The distribution patterns and characteristics of the microbiota in the reproductive tract of subjects were analyzed and were interpreted in relation to the clinical outcomes of the subjects.ResultsWhile the ovarian cancer was able to alter the genital microbiota, the bacteria were the dominant microorganisms in all samples across all cohorts in the study (median 99%). The microbiota of the upper female reproductive tract were mainly from the cervical, identified by low bacterial biomass and high bacterial diversity. Ovarian cancer had a distinct microbiota signature. The tubal ligation affects its microbial distribution. There were no different species on the surface of platinum-sensitive ovarian tissues compared to samples from platinum-resistant patients.ConclusionThe ovarian cancer–induced changes in microbiota magnify the potential of microbiota as a biotherapeutic modality in the treatment of ovarian cancer in this study and very likely for several malignancies and other conditions. Our findings demonstrated, for the first time, that microbiota could be dissected and applied in more specific fashion based on a predictive, preventive, and personalized medicine (PPPM) model in the treatment of ovarian cancer. Utilizing microbiota portfolio in a PPPM system in ovarian cancer would provide a unique opportunity to a clinically intelligent and novel approach in the treatment of ovarian cancer as well as several other conditions and malignancies.

Microbiome as a predictor of implantation.

Review the latest research on the female urogenital microbiome as a predictor of successful implantation. Lactobacillus crispatus seems to be beneficial species in a healthy female genital tract, although the presence of anaerobic bacteria and their impact has yet to be determined. The vaginal microbiome is associated with assisted reproductive technology (ART) outcome in terms of successful implantation and pregnancy. Approaches restoring a dysbiotic vaginal microbiome seem promising. It is questionable if a unique endometrial microbiome exists, given the low bacterial biomass, the invasiveness of endometrial sampling, and its associated high contamination risk. Future studies should focus on the whole microbiome using proteomics and metabolomics, as well as the virome to get a more holistic understanding of its role in reproduction. The vaginal and endometrial compartments are being studied to determine a healthy and unhealthy microbiome composition. Defining a healthy composition could provide insight into physiological processes related to the success of embryo implantation. The vaginal microbiome is easily accessible and its composition can be reliably assessed and can be associated with ART outcome. The existence of an endometrial or uterine microbiome is still debated, due to the combination of low biomass and unavoidable high risk of contamination during sampling.

SO-26 Identification and quantification of the microbiome in colorectal cancer metastases

Recent research has identified the existence of living bacteria in the tumor microenvironment, as well as cancer-specific bacteria in blood of patients. Early evidence suggested a role of the gut and tissue microbiome in the development of colorectal cancer (CRC). However, the part of this intra-tumor microbiome in promoting or aiding metastasis to distant organs remains unexplored. Interestingly, similar bacterial strains from CRC primary tumors have been identified in patient-matched liver metastases. Few mechanistic hypotheses have been proposed of how bacterial translocation to distant sites occurs, or whether the tumor-resident microbiome can influence the spreading or the behavior of metastases and their local immune response. We aimed to characterize the intra-tumor bacterial signature of metastatic colorectal cancer (mCRC) through a large-scale study with different cohorts from several biobanks in Belgium. We collected 378 frozen samples from 99 patients composed by 104 primary colorectal tumors (PT) and 99 patient-matched liver metastases (LMT), both associated with normal adjacent tissue (NAT) as control (n = 83 and 58, respectively). We added primary liver tumors (hepatocellular carcinoma (HCC, n=28) and cholangiocarcinoma (CGC, n=6)) from 27 patients as comparative cohort for LMT. The V4 region of the bacterial 16S rRNA gene was sequenced. The computational DADA2 pipeline was used to process the sequencing data and determine the abundance of amplicon sequence variants (ASV). We strictly applied sterile conditions from sample collection to sequencing, with negative and positive controls along the process to minimize contamination and misinterpretation of the results. Potential contaminants were removed bioinformatically by applying a series of stringent filters. Bacterial sequences were identified in all tissue types. Based on preliminary results, the principal component analysis based on bacterial composition of the samples revealed significant sample grouping (PERMANOVA, p-value = 1e-04) per tissue type (PT, LMT and HCC-CGC). The read number and bacterial composition of associated NAT were similar to their associated tumor tissue. PT, as expected by their colonic origin, presented higher bacterial biomass than LMT, yet the patient-specific microbiome composition in LMT was partially overlapping with PT, consisting with previously reported prevalent taxa. The number of shared ASVs between PT and LMT was significantly higher within than between patients (linear regression model with p-value = 4,22e-10), suggesting an individual bacterial transfer. This transfer appeared to be a probabilistic event, as it correlated with taxa abundance, and this transfer was more likely for the most abundant bacteria in the PT. Our preliminary results suggest the presence of a low bacterial biomass in LMT with characteristics closer to PT compared to primary tumors of the liver, suggesting a “per-tumor type” rather than “per-organ” bacterial signature and abundance. A within-patient bacterial transfer from PT to LMT seems to be observed for the most abundant taxa. These preliminary results are currently in a validation process. If our results are confirmed, this multicentric large-scale study would allow us to characterize and classify tumor tissue bacterial signature in mCRC to compare it with genomic and immune tumor features.

New Paradigms for Familiar Diseases: Lessons Learned on Circulatory Bacterial Signatures in Cardiometabolic Diseases.

Despite the strongly accumulating evidence for microbial signatures in metabolic tissues, including the blood, suggesting a novel paradigm for metabolic disease development, the notion of a core blood bacterial signature in health and disease remains a contentious concept. Recent studies clearly demonstrate that under a strict contamination-free environment, methods such as 16 S rRNA gene sequencing, fluorescence in-situ hybridization, transmission electron microscopy, and several more, allied with advanced bioinformatics tools, allow unambiguous detection and quantification of bacteria and bacterial DNA in human tissues. Bacterial load and compositional changes in the blood have been reported for numerous disease states, suggesting that bacteria and their components may partially induce systemic inflammation in cardiometabolic disease. This concept has been so far primarily based on measurements of surrogate parameters. It is now highly desirable to translate the current knowledge into diagnostic, prognostic, and therapeutic approaches.This review addresses the potential clinical relevance of a blood bacterial signature pertinent to cardiometabolic diseases and outcomes and new avenues for translational approaches. It discusses pitfalls related to research in low bacterial biomass while proposing mitigation strategies for future research and application approaches.

The bovine milk microbiome – an evolving science

Improved access to genome based, culture independent methods has generated great interest in defining the bovine milk microbiome. Several comprehensive reviews of this subject have recently been published and the purpose of this short review is to consolidate current understanding of the relevance and biological significance of this emerging topic. In contrast to mucosal organs that contain rich and well-characterized culturable and nonculturable microbial communities, milk obtained from the healthy bovine mammary gland usually contains few or no viable bacteria. The low bacterial biomass of milk has created methodological challenges that have resulted in considerable variability in results of studies that have used genomic methods to define the microbiota of milk obtained from healthy or diseased mammary glands. While genomes from several bacterial genera are routinely identified from samples of milk, teat skin and the teat canal, the viability, origin, and function of these organisms is uncertain as environmental factors have been shown to strongly influence the composition of these bacterial populations. Possible sources of microbial DNA include bacteria introduced from skin or the environment, bacteria trapped in teat canal keratin or bacteria engulfed by phagocytes. Researchers have not achieved consensus about key concepts such as the presence of a core commensal milk microbiome or dysbiosis as part of a causal pathway disrupting udder health. Understanding of the bovine milk microbiome has been greatly impeded by a lack of standardized methods used to collect, process, and assess bovine milk samples. Sample collection is a critical first step that will determine the validity of results. To minimize contamination with external sources of bacterial DNA, teat sanitation methods used for collection of milk samples that will be subjected to extraction and amplification of bacteria DNA should far exceed aseptic techniques used for collection of milk samples that will be submitted for microbiological culture. A number of laboratory issues have yet to be resolved. Contamination of low biomass samples with bacterial DNA from laboratory reagents is a well-known issue that has affected results of studies using bovine milk samples and results of sequencing of negative controls should always be reported. Replication of experiments has rarely been performed and consistency in results are lacking. While progress has been made, standardization of methods and replication using samples originating from differing farm conditions are critically needed to solidify knowledge of this emerging topic.

DdPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

BackgroundOne limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.ResultsUsing a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts.ConclusionOur improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

An investigation of upland soil fertility from different soil types

Soil type is a vital determinant of soil fertility because of its characteristic biological, chemical, and physical properties. However, the soil fertility of upland soil is probably changed by different agricultural management practices regardless of soil type. This study investigated the features of soil fertility (bacterial biomass, total carbon (TC), and total nitrogen (TN)) in upland fields in Japan. One thousand soil samples from different soil types were collected from upland fields located on 36 prefectures in Japan. The soil fertility was analyzed using the Soil Fertility Index (SOFIX). There were six soil types included in this study: Organic soils (B), Andosols (D), Lowland soils (F), Red-yellow soils (G), Stagnic soils (H), and Brown Forest soils (I). Of these, the soil types D and F occupied the largest percentage of samples. The values of bacterial biomass, TC, and TN varied greatly, regardless of soil type. The soil fertility does not seem to be characterized by the soil type in upland soils in Japan. The correlations between bacterial biomass and TC (r = 0.23, p < 0.01) and bacterial biomass and TN (r = 0.16, p < 0.01) were relatively weak. Conversely, the correlation between TC and TN (r = 0.68, p < 0.01) was moderate. In soil type D, up to 40% of samples had no bacterial biomass detected and 17% possessed a low bacterial biomass. The effect of the soil types may not be a critical factor in soil fertility in the upland fields.

How the 'kitome' influences the characterization of bacterial communities in lepidopteran samples with low bacterial biomass.

We aimed to elucidate whether the DNA extraction kit and bacteria therein affect the characterization of bacterial communities associated with butterfly samples harbouring different bacterial abundancies. We analysed bacteria associated with eggs of Pieris brassicae and with adults of this butterfly, which were either untreated or treated with antibiotics (ABs). Three DNA extraction kits were used. Regardless of the extraction kit used, PCR amplification of the bacterial 16S rRNA gene detected very low bacterial presence in eggs and AB-treated butterflies. In untreated butterflies, bacterial signal intensity varied according to the kit and primers used. Sequencing (MiSeq) of the bacterial communities in untreated and AB-treated butterflies revealed a low alpha diversity in untreated butterflies because of the dominance of few bacteria genera, which were detectable regardless of the kit. However, a significantly greater alpha diversity was found in AB-treated butterflies, evidencing a true bias of the results due to bacterial contaminants in the kit. The so-called 'kitome' can impact the profiling of Lepidoptera-associated bacteria in samples with low bacterial biomass. Our study highlights the necessity of method testing and analysis of negative controls when investigating Lepidoptera-associated bacterial communities.

Placentas delivered by pre-pregnant obese women have reduced abundance and diversity in the microbiome.

Maternal pre‐pregnancy obesity may have an impact on both maternal and fetal health. We examined the microbiome recovered from placentas in a multi‐ethnic maternal pre‐pregnant obesity cohort, through an optimized microbiome protocol to enrich low bacterial biomass samples. We found that the microbiomes recovered from the placentas of obese pre‐pregnant mothers are less abundant and less diverse when compared to those from mothers of normal pre‐pregnancy weight. Microbiome richness also decreases from the maternal side to the fetal side, demonstrating heterogeneity by geolocation within the placenta. In summary, our study shows that the microbiomes recovered from the placentas are associated with pre‐pregnancy obesity.ImportanceMaternal pre‐pregnancy obesity may have an impact on both maternal and fetal health. The placenta is an important organ at the interface of the mother and fetus, and supplies nutrients to the fetus. We report that the microbiomes enriched from the placentas of obese pre‐pregnant mothers are less abundant and less diverse when compared to those from mothers of normal pre‐pregnancy weight. More over, the microbiomes also vary by geolocation within the placenta.

Current ocular microbiome investigations limit reproducibility and reliability: Critical review and opportunities

Enthusiasm for research describing microbial communities using next-generation sequencing (NGS) has outpaced efforts to standardize methodology. Without consistency in the way research is carried out in this field, the comparison of data between studies is near impossible and the utility of results remains limited. This holds true for bacterial microbiome research of the ocular surface, and other sites, in both humans and animals. In addition, the ocular surface remains under-explored when compared to other mucosal sites. Low bacterial biomass samples from the ocular surface lead to further technical challenges. Taken together, two major problems were identified: (1) Normalization of the workflow in studies utilizing NGS to investigate the ocular surface bacteriome is necessary in order to propel the field forward and improve research impact through cross-study comparisons. (2) Current microbiome profiling technology was developed for high bacterial biomass samples (such as feces or soil), posing a challenge for analyses of samples with low bacterial load such as the ocular surface. This article reviews the challenges and limitations currently facing ocular microbiome research and provides recommendations for minimum reporting standards for veterinary ophthalmologists and clinician scientists to limit inter-study variation, improve reproducibility, and ultimately render results from these studies more impactful. The move toward normalization of methodology will expedite and maximize the potential for microbiome research to translate into meaningful discovery and tangible clinical applications.

Protoblock - A biological standard for formalin fixed samples

BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissue is the gold standard in pathology tissue storage, representing the largest collections of patient material. Their reliable use for DNA analyses could open a trove of potential samples for research and are currently being recognised as a viable source material for bacterial analysis. There are several key features which limit bacterial-related data generation from this material: (i) DNA damage inherent to the fixing process, (ii) low bacterial biomass that increases the vulnerability to contamination and exacerbates the host DNA effects and (iii) lack of suitable DNA extraction methods, leading to data bias. The development and systematic use of reliable standards is a key priority for microbiome research. More than perhaps any other sample type, FFPE material urgently requires the development of standards to ensure the validity of results and to promote reproducibility.ResultsTo address these limitations and concerns, we have developed the Protoblock as a biological standard for FFPE tissue-based research and method optimisation. This is a novel system designed to generate bespoke mock FFPE ‘blocks’ with a cell content that is user-defined and which undergoes the same treatment conditions as clinical FFPE tissues. The ‘Protoblock’ features a mix of formalin-fixed cells, of known number, embedded in an agar matrix which is solidified to form a defined shape that is paraffin embedded.The contents of various Protoblocks populated with mammalian and bacterial cells were verified by microscopy. The quantity and condition of DNA purified from blocks was evaluated by qPCR, 16S rRNA gene amplicon sequencing and whole genome sequencing. These analyses validated the capability of the Protoblock system to determine the extent to which each of the three stated confounding features impacts on eventual analysis of cellular DNA present in FFPE samples.ConclusionThe Protoblock provides a representation of biological material after FFPE treatment. Use of this standard will greatly assist the stratification of biological variations detected into those legitimately resulting from experimental conditions, and those that are artefacts of the processed nature of the samples, thus enabling users to relate the outputs of laboratory analyses to reality.BKi5aTsaSPPC22FkaWVr1zVideo