Pancreatic cancers have high mortality and rising incidence rates which may be related to unhealthy western-type dietary and lifestyle patterns as well as increasing body weights and obesity rates. Recent data also suggest a role for the gut microbiome in the development of pancreatic cancer. Here, we review the experimental and observational evidence for the roles of the oral, gut and intratumoural microbiomes, impaired gut barrier function and exposure to inflammatory compounds as well as metabolic dysfunction as contributors to pancreatic disease with a focus on pancreatic ductal adenocarcinoma (PDAC) initiation and progression. We also highlight some emerging gut microbiome editing techniques currently being investigated in the context of pancreatic disease. Notably, while the gut microbiome is significantly altered in PDAC and its precursor diseases, its utility as a diagnostic and prognostic tool is hindered by a lack of reproducibility and the potential for reverse causality in case-control cohorts. Future research should emphasise longitudinal and mechanistic studies as well as integrating lifestyle exposure and multi-omics data to unravel complex host-microbiome interactions. This will allow for deeper aetiologic and mechanistic insights that can inform treatments and guide public health recommendations.

Studies in humans and large animals indicate a relationship between the uterine microbiome composition and endometrial receptivity. Despite many studies have been performed, the analysis of the uterine microbiome remains challenging due to the very low microbial biomass. Studies in other biological systems showed that RNA-based microbiome analysis complements DNA-based results and provides information about active bacteria in a sample. Thus, the aim of this study was to establish a highly sensitive and specific 16S rRNA gene V3-V4 amplicon PCR from equine uterine cytobrush samples and to compare DNA- and RNA-based 16S rRNA microbiome analysis. An optimized 16S rRNA gene V3-V4 amplicon PCR protocol from equine uterine cytobrush samples was developed, which was able to detect less than 38 bacterial genome copies using a bacterial DNA community standard. For the RNA-based amplicon generation protocol starting from cDNA, at least a 10-fold higher sensitivity was estimated compared to DNA-based approach. The comparison of using RNA and DNA isolated from the same uterine cytobrush samples as input for 16S V3-V4 amplicon sequencing revealed a much higher number of amplicon sequence variants as well as taxonomic units for the RNA-based approach. This resulted in significant differences in alpha (Simpson, Chao1) and beta diversity between RNA- and DNA-based analysis. Differential abundance analysis revealed significant differences between DNA and RNA samples at all taxonomic levels. Despite these differences, the overall microbiome composition was similar between the paired DNA and RNA samples. Many differences were probably found due to the higher sensitivity of the RNA-based approach. Furthermore, the DNA-based analysis is biased by the rRNA gene copy numbers (1-21), and the RNA-based analysis by the number of ribosomes per cell, which was reflected in the differences in the microbiome composition between the approaches. In addition, the results suggested that the DNA-based analysis is detecting cell-free bacterial DNA and/or DNA of dead bacteria that could be present in the samples. Altogether, the obtained results indicate advantages of a combined DNA- and RNA-based microbiome analysis, offering complementary and valuable information in the context of fertility-related studies of the uterine microbiome.

Metagenomics, the genomic analysis of all species present within a mixed population, is an important tool used for the exploration of microbiomes in clinical and environmental microbiology. Whilst the development of next-generation sequencing, and more recently third generation long-read approaches such as nanopore sequencing, have greatly advanced the study of metagenomics, recovery of unbiased material from microbial populations remains challenging. One promising advancement in genomic sequencing from Oxford Nanopore Technologies (ONT) is adaptive sampling, which enables real-time enrichment or depletion of target sequences. As sequencing technologies continue to develop, and advances such as adaptive sampling become common techniques within the microbiological toolkit, it is essential to evaluate the benefits of such advancements to metagenomic studies, and the impact of methodological choices on research outcomes.

Given the rapid development of sequencing tools and chemistry, this study aimed to demonstrate the impacts of choice of DNA extraction kit and sequencing chemistry on downstream metagenomic analyses. We first explored the quality and accuracy of 16S rRNA amplicon sequencing for DNA extracted from the ZymoBIOMICS Microbial Community Standard, using a range of commercially available DNA extraction kits to understand the effects of different kit biases on assessment of microbiome composition. We next compared the quality and accuracy of metagenomic analyses for two nanopore-based ligation chemistry kits with differing levels of base-calling error; the older and more error-prone (~ 97% accuracy) LSK109 chemistry, and newer more accurate (~ 99% accuracy) LSK112 Q20 + chemistry. Finally, we assessed the impact of the nanopore sequencing chemistry version on the output of the novel adaptive sampling approach for real-time enrichment of the genome for the yeast Saccharomyces cerevisiae from the microbial community.

Firstly, DNA extraction kit methodology impacted the composition of the yield, with mechanical bead-beating methodologies providing the least biased picture due to efficient lysis of Gram-positive microbes present in the community standard, with differences in bead-beating methodologies also producing variation in composition. Secondly, whilst use of the Q20 + nanopore sequencing kit chemistry improved the base-calling data quality, the resulting metagenomic assemblies were not significantly improved based on common metrics and assembly statistics. Most importantly, we demonstrated the effective application of adaptive sampling for enriching a low-abundance genome within a metagenomic sample. This resulted in a 5-7-fold increase in target enrichment compared to non-adaptive sequencing, despite a reduction in overall sequencing throughput due to strand-rejection processes. Interestingly, no significant differences in adaptive sampling enrichment efficiency were observed between the older and newer ONT sequencing chemistries, suggesting that adaptive sampling performs consistently across different library preparation kits.

Our findings underscore the importance of selecting a DNA extraction methodology that minimises bias to ensure an accurate representation of microbial diversity in metagenomic studies. Additionally, despite the improved base-calling accuracy provided by newer Q20 + sequencing chemistry, we demonstrate that even older ONT sequencing chemistries can achieve reliable metagenomic sequencing results, enabling researchers to confidently use these approaches depending on their specific experimental needs. Critically, we highlight the significant potential of ONT's adaptive sampling technology for targeted enrichment of specific genomes within metagenomic samples. This approach offers broad applicability for enriching target organisms or genetic elements (e.g., pathogens or plasmids) or depleting unwanted DNA (e.g., host DNA) in diverse sample types from environmental and clinical studies. However, researchers should carefully weigh the benefits of adaptive sampling against the potential trade-offs in sequencing throughput, particularly for low-abundance targets, where strand rejection can lead to pore blocking. These results provide valuable guidance for optimising adaptive sampling in metagenomic workflows to achieve specific research objectives.

Bacteria are the major component of poultry gastrointestinal tract (GIT) microbiota and play an important role in host health, nutrition, physiology regulation, intestinal development, and growth. Bacterial community profiling based on the 16S ribosomal RNA (rRNA) gene amplicon sequencing approach has become the most popular method to determine the taxonomic composition and diversity of the poultry microbiota. The 16S rRNA gene profiling involves numerous steps, including sample collection and storage, DNA isolation, 16S rRNA gene primer selection, Polymerase Chain Reaction (PCR), library preparation, sequencing, raw sequencing reads processing, taxonomic classification, α- and β-diversity calculations, and statistical analysis. However, there is currently no standardized protocol for 16S rRNA gene analysis profiling and data deposition for poultry microbiota studies. Variations in DNA storage and isolation, primer design, and library preparation are known to introduce biases, affecting community structure and microbial population analysis leading to over- or under-representation of individual bacteria within communities. Additionally, different sequencing platforms, bioinformatics pipeline, and taxonomic database selection can affect classification and determination of the microbial taxa. Moreover, detailed experimental design and DNA processing and sequencing methods are often inadequately reported in poultry 16S rRNA gene sequencing studies. Consequently, poultry microbiota results are often difficult to reproduce and compare across studies. This manuscript reviews current practices in profiling poultry microbiota using 16S rRNA gene amplicon sequencing and proposes the development of guidelines for protocol for 16S rRNA gene sequencing that spans from sample collection through data deposition to achieve more reliable data comparisons across studies and allow for comparisons and/or interpretations of poultry studies conducted worldwide.

Research on insect-microbe relationships is booming, with DNA sequencing being the most commonly used method to describe insect microbiota. However, sequencing is vulnerable to contamination, especially when the sample has low microbial biomass. Such low-biomass samples are common across insect taxa, developmental stages, and tissue types. Identifying putative contaminants is essential to distinguish between true microbiota and introduced contaminant DNA. It is therefore important that studies control for contamination, but how often this is done is unknown. To investigate the status quo of contamination control, we undertook a systematic literature review to quantify the prevalence of negative control usage and contamination control across the literature on insect microbiota (specifically bacterial communities) over a 10 year period. Two-thirds of the 243 insect microbiota studies evaluated had not included blanks (negative controls), and only 13.6% of the studies sequenced these blanks and controlled for contamination in their samples. Our findings highlight a major lack of contamination control in the field of insect microbiota research. This result suggests that a number of microbes reported in the literature may be contaminants as opposed to insect-associated microbiota and that more rigorous contamination control is needed to improve research reliability, validity, and reproducibility. Based on our findings, we recommend the previously developed guidelines outlined in the RIDE checklist, with the addition of one more guideline. We refer to this as the RIDES checklist, which stands for Report methodology, Include negative controls, Determine the level of contamination, Explore contamination downstream, and State the amount of off-target amplification.IMPORTANCEOur systematic review reveals a major lack of methodological rigor within the field of research on insect-associated microbiota. The small percentage of studies that control for contamination suggests that an unknown but potentially considerable number of bacteria reported in the literature could be contaminants. The implication of this finding is that true microbiota may be masked or misrepresented, especially in insects with low microbial biomass.

The microbiome remains a top area of research, and it is now common to examine any organic and inorganic samples for bacterial colonization. However, due to the ubiquity of bacteria in the environment, separating the low-burden colonization of bacteria from the possible contamination of laboratory reagents remains problematic. When examining samples of expected low bacterial burden, it is common to first amplify any bacterial DNA present through PCR before sequencing. In this work, we examined nine different commercial PCR enzymes and their reaction components as possible sources of bacterial DNA contamination. We found contaminating bacterial DNA in seven of the nine reactions, and this DNA was shown to come from a variety of species. Importantly, we were able to perform these studies solely with endpoint PCR and Sanger sequencing, which are more accessible and affordable than high-throughput, short-read sequencing and real-time PCR. This work confirms that there needs to be an increased emphasis on including control reactions in microbiome studies so that contaminating DNA sequences can be identified and addressed, and that this can be achieved with minimal resources.

The microbiota has been unequivocally linked to various diseases, yet the mechanisms underlying these associations remain incompletely understood. One potential contributor to this relationship is the extracellular vesicles produced by bacteria (bEVs). However, the detection of these bEVs is challenging. Therefore, we propose a novel workflow to identify bacterial RNA present in circulating extracellular vesicles using Total EV RNA-seq data. As a proof of concept, we applied this workflow to a dataset from individuals with multiple sclerosis (MS).

We analyzed total EV RNA-seq data from blood samples of healthy controls and individuals with MS, encompassing both the Relapsing-Remitting (RR) and Secondary Progressive (SP) phases of the disease. Our workflow incorporates multiple reference mapping steps against the host genome, followed by a consensus selection of bacterial genera based on various taxonomic profiling tools. This consensus approach utilizes a flagging system to exclude genera with low abundance across profilers. Additionally, we included EVs derived from two cultured species that serve as biological controls, as well as artificially generated reads from 60 species as a technical control, to validate the specificity of this workflow.

Our findings demonstrate that bacterial RNA can indeed be detected in total EV RNA-seq from blood samples, suggesting that this workflow can be a powerful tool for reanalyzing RNA-seq data from EV studies. Additionally, we identified promising bacterial candidates with differential expression between the RR and SP phases of MS.

This approach provides valuable insights into the potential role of bEVs in the microbiota-host communication. Finally, this approach is translatable to other experiments using total RNA, where the lack of a robust pipeline can lead to an increased false positive detection of microbial genera. The workflow and instructions on how to use it are available at the following repository: https://github.com/NanoNeuro/EV_taxprofiling.

The upper and lower respiratory tracts feature distinct environments and responses affecting microbial colonization but investigating the relationship between them is technically challenging. We aimed to identify relationships between taxa colonizing the nasopharynx and trachea across childhood.

We employed V4 16S rRNA gene sequencing to profile nasopharyngeal swabs and tracheal aspirates collected from 172 subjects between 20 weeks and 18 years of age. These samples were collected prior to elective procedures over the course of 20 weeks in 2020 from subjects enrolled in a cross-sectional study. After extraction, sequencing, and quality control, we studied the remaining 147 of 172 nasopharyngeal swabs and 95 of 172 tracheal aspirates, including 80 subject-matched pairs of samples.

Sequencing data revealed that the nasopharynx is colonized by few, often highly abundant taxa, while the tracheal aspirates feature greater diversity. The patterns of colonization identified in the nasopharynx correlate with subject age across childhood.

Our data suggests that there are relatively few species that colonize both the nasopharyngeal tract and the trachea. Furthermore, we observe a pattern of change in the nasopharyngeal microbiota that is correlated with age, suggesting a possible developmental progression of the nasopharyngeal microbiota across childhood.

The airway microbiota in childhood plays important roles in respiratory health and immune development. In this work, we report on paired nasopharyngeal swab and tracheal aspirate samples from a cross-sectional cohort of children from infancy to 18 years. We find that the upper and lower airway microbiota are unlikely to share taxa and do not correlate in terms of diversity. We show that the composition of the upper airway microbiota is strongly correlated with age, with a stereotypic developmental trajectory during childhood and adolescence. Our results inform our understanding of airway microbiota assembly and may be used to predict airway disease in young children.

The goal of this study was to characterize the microbial profile of two different fresh pork cuts, bootjack (BJ) trim and tenderloin (TL), through a 16S rRNA sequencing workflow developed specifically for investigating low-biomass fresh meat within a commercial production schedule. Additionally, this study aimed to determine a baseline Salmonella prevalence and enumeration profile across these two fresh pork cuts. Results showed that microbiome diversity was different between the BJ and TL, and also differed significantly by processing date. The relative abundance of key bacterial genera associated with food safety and spoilage was also different between the two meat types. However, over the course of the production shift, changes in the meat microbiome were limited in both the BJ and TL. The crude prevalence and enumerated burden of Salmonella were lower than what has been previously reported in similar fresh pork cuts, and all of the Salmonella-positive samples occurred on just two processing windows of 1-2 days each. Taken together, the results of this study suggest that the microbial profile of two fresh pork cuts is significantly different even within the same plant at the same time points, and that day-to-day variability within the production process likely influences both the fresh pork microbiome and Salmonella profile of these two meat types.IMPORTANCEModern pork processing involves a series of processes that begin with the handling and transport of the live animals, proceed through harvest and fabrication, and end with the packaging and distribution of fresh pork to the consumer. Each step in this process can alter the microbial community of fresh pork and influence the meat's safety and shelf life. However, little is known about the microbial ecology of individual, unprocessed pork cuts and if the diversity of the meat microbiome remains consistent throughout a production schedule. Additionally, the crude prevalence and enumeration of Salmonella have not been well established for individual fresh pork cuts throughout a production schedule. A more thorough understanding of the microbial profile at different stages of pork production will help processors determine processing steps that impact the microbial characteristics of fresh pork. This insight will help processors implement targeted intervention strategies to enhance food safety and quality.

The advent of next-generation sequencing (NGS) technologies has made it possible to investigate microbial communities in various environments, including different sites within the human body. Therefore, the previously established belief of the sterile nature of several body sites, including human blood, has now been challenged. However, metagenomics investigation of areas with an anticipated low microbial biomass may be susceptible to misinterpretation. Here, we critically evaluate the results of 16S targeted amplicon sequencing performed on total DNA collected from healthy donors' blood samples while incorporating specific negative controls aimed at addressing potential bias to supplement and strengthen the research in this area. We prepared negative controls by increasing the initial DNA quantity through sequences that can be recognized and subsequently discarded. We found that only three organisms were sporadically present among the samples, and this was mostly attributable to bacteria ubiquitously present in laboratory reagents. Despite not fully confirming or denying the existence of healthy blood microbiota, our results suggest that living bacteria, or at least their residual DNA sequences, are not a common feature of human blood in healthy people. Finally, our study poses relevant questions on the design of controls in this research area that must be considered in order to avoid misinterpreted results that appear to contaminate current high-throughput research.

Antimicrobial resistance poses a significant threat to global health, with its spread intricately linked across human, animal, and environmental sectors. Revealing the antimicrobial resistance gene (ARG) flow among the One Health sectors is essential for better control of antimicrobial resistance.

In this study, we investigated regional ARG transmission among humans, food, and the environment in Dengfeng, Henan Province, China by combining large-scale metagenomic sequencing with culturing of resistant bacterial isolates in 592 samples. A total of 40 ARG types and 743 ARG subtypes were identified, with a predominance of multidrug resistance genes. Compared with microbes from human fecal samples, those from food and environmental samples showed a significantly higher load of ARGs. We revealed that dietary habits and occupational exposure significantly affect ARG abundance. Pseudomonadota, particularly Enterobacteriaceae, were identified as the main ARG carriers shaping the resistome. The resistome in food samples was found more affected by mobile genetic elements (MGEs), whereas in environmental samples, it was more associated with the microbial composition. We evidenced that horizontal gene transfer (HGT) mediated by plasmids and phages, together with strain transmission, particularly those associated with the Enterobacteriaceae members, drive regional ARG flow. Lifestyle, dietary habits, and occupational exposure are all correlated with ARG dissemination and flies and food are important potential sources of ARGs to humans. The widespread mobile carbapenemase gene, OXA-347, carried by non-Enterobacteriaceae bacteria in the human gut microbiota, requires particular attention. Finally, we showed that machine learning models based on microbiome profiles were effective in predicting the presence of carbapenem-resistant strains, suggesting a valuable approach for AMR surveillance.

Our study provides a full picture of regional ARG transmission among the One Health sectors in a county-level city in China, which facilitates a better understanding of the complex routes of ARG transmission and highlights new points of focus for AMR surveillance and control. Video Abstract.

Archaea have been identified as early colonizers of the human intestine, appearing from the first days of life. It is hypothesized that the origin of many of these archaea is through vertical transmission during breastfeeding. In this study, we aimed to characterize the archaeal composition in samples of mother-neonate pairs to observe the potential vertical transmission. We performed a cross-sectional study characterizing the archaeal diversity of 40 human colostrum-neonatal stool samples by next-generation sequencing of V5-V6 16S rDNA libraries. Intra- and inter-sample analyses were carried out to describe the Archaeal diversity in each sample type. Human colostrum and neonatal stools presented similar core microbiota, mainly composed of the methanogens Methanoculleus and Methanosarcina. Beta diversity and metabolic prediction results suggest homogeneity between sample types. Further, the co-occurrence network analysis showed associations between Archaea and Bacteria, which might be relevant for these organisms' presence in the human milk and neonatal stool ecosystems. According to relative abundance proportions, beta diversity, and co-occurrence analyses, the similarities found imply that there is vertical transmission of archaea through breastfeeding. Nonetheless, differential abundances between the sample types suggest other relevant sources for colonizing archaea to the neonatal gut.

The factors influencing meconium aspiration syndrome (MAS) severity remain poorly understood. In a piglet model of MAS, we hypothesized the respiratory microbiome would reflect the bacterial signature of meconium with short-chain fatty acid (SCFA) accumulation as a byproduct of bacterial fermentation.

Cesarean section at approximately 115-day term was performed on two sows. Male (9) and female (3) piglets were delivered, instrumented, anesthetized, and randomized into a Control (n = 6) or MAS group (n = 6). MAS received a meconium slurry (3 mL/kg) aspiration injury. Experimental animals were monitored continuously, ventilated, and resuscitated for 24 h. BALF was collected for 16S microbiome sequencing and SCFA analysis by gas chromatography. Effects of SCFAs on A549 alveolar pulmonary epithelial in vitro cell viability and inflammation were assessed.

The MAS group had significantly higher fluid and vasopressor requirements than the Control group (p < 0.05) though both groups developed lung injury. The meconium microbiome demonstrated a difference in genus proportions as compared with the BALF of the Control and MAS groups. The MAS group had a relative increase in propionic acid-forming bacteria and higher BALF concentrations of propionic acid (0.6 ± 0.2 mmol/kg) than the Control group (0.2 ± 0.2 mmol/kg; p > 0.05). Propionic acid was associated with decreased pulmonary epithelial cell viability and an upregulated pro-inflammatory response.

Meconium may host a microbiome with byproducts that interact with the pulmonary epithelium and influence lung injury severity in MAS.

This review examines the longstanding debate of nature and intrauterine environmental challenges that shapes human development and behavior, with a special focus on the influence of maternal prenatal gut microbes. Recent research has revealed the critical role of the gut microbiome in human neurodevelopment, and evidence suggest that maternal microbiota can impact fetal gene and microenvironment composition, as well as immunophysiology and neurochemical responses. Furthermore, intrauterine neuroepigenetic regulation may be influenced by maternal microbiota, capable of having long-lasting effects on offspring behavior and cognition. By examining the complex relationship between maternal prenatal gut microbes and human development, this review highlights the importance of early-life environmental factors in shaping neurodevelopment and cognition.

Microbiome studies targeting hypervariable regions of the 16S rRNA gene are suitable for understanding interactions between animals and their associated bacteria. While many studies focus on the gut microbiome, assessments of blood microbiota remain scarce despite the prevalence of blood-borne pathogens in vertebrates. This study aimed to investigate the bacterial community in blood samples from 79 living and 7 road-killed lowland tapirs (Tapirus terrestris), a vulnerable species, sampled in two biomes in midwestern Brazil: Pantanal and Cerrado. Animals were categorized by condition (living or road-killed), sex, age, and biome. V3-V4 16S rRNA fragments were obtained from 86 blood samples and 4 negative controls. After filtering contaminants, 13,742,198 sequences representing 2146 ASVs were analyzed. Alpha diversity significantly differed by condition, while beta diversity differed by condition, site, and age (adults vs. sub-adults). For living animals (79/86 samples), alpha diversity showed no significant differences, but beta diversity differed by age. Different vector-borne bacterial pathogens, including Anaplasmataceae, Bartonella, and Borrelia spp., were detected. Additionally, evidence of transient translocation of microbial communities from other body regions to the bloodstream was observed. Amplification of bacterial 16S rRNA from blood samples of wild T. terrestris provided novel information about the diversity of blood-borne microbiota of lowland tapirs, members of a poorly studied mammalian family. Next-generation sequencing proved to be a valuable tool for screening potential vector-borne pathogens in this host.

Despite many decades of research, the exact etiology of pre-eclampsia (PE) remains unknown. Several etiopathologies have been suggested, including the role of the placental microbiota. However, the existence of placental microbiota and its possible contribution to pregnancy complications, particularly PE has remained controversial.

The present study was designed to identify different microbes that co-exist the placenta of women with early- and late-onset PE.

Thirty age-matched normotensive and early-onset as well as age-matched normotensive and late-onset pre-eclamptic women respectively, were recruited. After obtaining an informed consent, the placental tissues were obtained through caesarian section with sterile and standardized clinical procedures. DNA was extracted from each tissue and microbiome analysis was conducted using a targeted 16 S analysis and the reads were analyzed with bioinformatics.

There was a significance difference between the blood pressure of early-/late-onset PE compared with age-matched normotensive controls, respectively. In addition, the reads from placencental samples were classified as belonging to the phyla, Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria, with Proteobacteria dominated by the classes Pseudomonadales and Gammaproteobacteria with smaller amounts of Actinobacteria and Bacteroidetes. There was no significant difference between the placental bacterial species of early-/late-onset PE compared with age-matched normotensive controls, respectively. Further analysis found no correlation between bacterial species and early- or late-onset PE.

The present results demonstrate a low biomass of bacterial species, which might further indicate that the placental samples had very low levels of bacteria species and there is no correlation between the bacterial composition and early- or late-onset PE.

Cockles play a vital ecological role and provide valuable ecosystem services globally. However, the performance, production, and safe consumption of cockles are significantly influenced by their gut-associated bacteriome. Accurate understanding of gut-bacteriome interactions, and surveillance of pathogenic bacteria loads in cockles, rely on efficient DNA extraction methods that yield high-quality and representative bacterial DNA. Despite this importance, reliable extraction methods for cockles are currently overlooked. Therefore, we evaluated the performance of five DNA extraction kits (E.Z.N.A.® Soil DNA; FastDNA® Spin; DNeasy PowerSoil Pro; QIAamp PowerFecal DNA; ZymoBIOMICS™DNA Miniprep) in terms of DNA quality, yield, bacterial community structure (analysed by using denaturating gradient gel electrophoresis; DGGE), and bacteriome composition (analysed by 16S rRNA gene sequencing) in Cerastoderma edule gut. The DNeasy kit provided the highest purity and quantity of bacterial DNA, while the PowerFecal and Zymo kits exhibited reduced extraction efficiency. DGGE profiles revealed significant variability between the tested kits (R = 0.512; mean P = 0.011), but the FastDNA kit under-represented the bacterial community in cockles' gut. Based on alpha diversity, the DNeasy kit outperformed the others and successfully detected all abundant genera found with the alternative kits. Our findings indicate that the DNeasy kit is an efficient DNA extraction method, enabling a molecular representation of the gut-associated bacteriome in C. edule. These results contribute to the development of effective techniques for studying the cockle gut bacteriome and its ecological implications.

Numerous studies have shown that a healthy reproductive tract microbiota is crucial for successful reproduction and that its composition is influenced by various environmental and host factors. However, it is not known whether the reproductive microbiota is also shaped by the major histocompatibility complex (MHC), a family of genes essential to differentiate 'self' from 'non-self' peptides to initiate an adaptive immune response. We tested the association between the follicular fluid microbiome and MHC genes in 27 women. Women with higher MHC diversity had a higher microbiome diversity, characterized by bacteria commonly associated with vaginal dysbiosis. Women with similar MHC genes were also similar in their microbiome composition, indicating that MHC composition may be a key factor in determining the bacterial assemblage in the reproductive tract. Finally, the composition of the follicular fluid microbiome was similar to the vaginal microbiome, suggesting that numerous bacteria of the vagina are true inhabitants of the follicular fluid or that vaginal microbiota contaminated the follicular fluid microbiota during transvaginal collection. Collectively, our results demonstrate the importance of host genetic factors in shaping women's reproductive microbiota and they open the door for further research on the role of microbiota in mediating MHC-related variation in reproductive success.

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10-9 ng/µl) and S. salivarius (2.5 ×10-6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10-4 ng/µl) and 0.48 copies/µl (2.5 ×10-7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.

As investigations of low-biomass microbial communities have become more common, so too has the recognition of major challenges affecting these analyses. These challenges have been shown to compromise biological conclusions and have contributed to several controversies. Here, we review some of the most common and influential challenges in low-biomass microbiome research. We highlight key approaches to alleviate these potential pitfalls, combining experimental planning strategies and data analysis methods.

Since the advent of next-generation sequencing, investigators worldwide have sought to discern whether a functional and biologically or clinically relevant prenatal microbiome exists. One line of research has led to the hypothesis that microbial DNA detected in utero/in ovo or prior to birth/hatching is a result of contamination and does not belong to viable and functional microbes. Many of these preliminary evaluations have been conducted in humans, mice, and nonhuman primates due to sample and specimen availability. However, a comprehensive review of the literature across animal species suggests organisms that maintain an obligate relationship with microbes may act as better models for interrogating the selective pressures placed on vertical microbial transfer over traditional laboratory species. To date, studies in humans and viviparous laboratory species have failed to illustrate the clear presence and transfer of functional microbes in utero. Until a ground truth regarding the status and relevance of prenatal microbes can be ascertained, it is salient to conduct parallel investigations into the prevalence of a functional prenatal microbiome across the developmental lifespan of multiple organisms in the kingdom Animalia. This comprehensive understanding is necessary not only to determine the role of vertically transmitted microbes and their products in early human health but also to understand their full One Health impact. This review is among the first to compile such comprehensive primary conclusions from the original investigator's conclusions, and hence collectively illustrates that prenatal microbial transfer is supported by experimental evidence arising from over a long and rigorous scientific history encompassing a breadth of species from kingdom Animalia.

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.

Patients with pancreatic ductal adenocarcinoma (PDAC) display an altered oral, gastrointestinal, and intra-pancreatic microbiome compared to healthy individuals. However, knowledge regarding the bile microbiome and its potential impact on progression-free survival in PDACs remains limited.

Patients with PDAC (n = 45), including 20 matched pairs before and after surgery, and benign controls (n = 16) were included prospectively. The characteristics of the microbiomes of the total 81 bile were revealed by 16  S-rRNA gene sequencing. PDAC patients were divided into distinct groups based on tumor marker levels, disease staging, before and after surgery, as well as progression free survival (PFS) for further analysis. Disease diagnostic model was formulated utilizing the random forest algorithm.

PDAC patients harbor a unique and diverse bile microbiome (PCoA, weighted Unifrac, p = 0.038), and the increasing microbial diversity is correlated with dysbiosis according to key microbes and microbial functions. Aliihoeflea emerged as the genus displaying the most significant alteration among two groups (p < 0.01). Significant differences were found in beta diversity of the bile microbiome between long-term PFS and short-term PFS groups (PCoA, weighted Unifrac, p = 0.005). Bacillota and Actinomycetota were identified as altered phylum between two groups associated with progression-free survival in all PDAC patients. Additionally, we identified three biomarkers as the most suitable set for the random forest model, which indicated a significantly elevated likelihood of disease occurrence in the PDAC group (p < 0.0001). The area under the receiver operating characteristic (ROC) curve reached 80.8% with a 95% confidence interval ranging from 55.0 to 100%. Due to the scarcity of bile samples, we were unable to conduct further external verification.

PDAC is characterized by an altered microbiome of bile ducts. Biliary dysbiosis is linked with progression-free survival in all PDACs. This study revealed the alteration of the bile microbiome in PDACs and successfully developed a diagnostic model for PDAC.

The etiology of pediatric acute lymphatic leukemia (ALL) is still unclear. Whole-metagenome shotgun sequencing of bone marrow samples in patients with treatment-naïve ALL (n=6) was performed for untargeted investigation of bacterial and viral DNA. The control group consisted of healthy children (n=4) and children with non-oncologic diseases (n=2) undergoing bone marrow sampling. Peripheral blood of all participants was investigated at the same time. After bioinformatical elimination of potential contaminants by comparison with the employed controls, no significant amounts of microbial or viral DNA were identified.

This study aims to validate the existence of a microbiome within intraductal papillary mucinous neoplasm (IPMN) that can be differentiated from the taxonomically diverse DNA background of next-generation sequencing procedures.

We generated 16S rRNA amplicon sequencing data to analyse 338 cyst fluid samples from 190 patients and 19 negative controls, the latter collected directly from sterile syringes in the operating room. A subset of samples (n=20) and blanks (n=5) were spiked with known concentrations of bacterial cells alien to the human microbiome to infer absolute abundances of microbial traces. All cyst fluid samples were obtained intraoperatively and included IPMNs with various degrees of dysplasia as well as other cystic neoplasms. Follow-up culturing experiments were conducted to assess bacterial growth for microbiologically significant signals.

Microbiome signatures of cyst fluid samples were inseparable from those of negative controls, with no difference in taxonomic diversity, and microbial community composition. In a patient subgroup that had recently undergone invasive procedures, a bacterial signal was evident. This outlier signal was not characterised by higher taxonomic diversity but by an increased dominance index of a gut-associated microbe, leading to lower taxonomic evenness compared with the background signal.

The 'microbiome' of IPMNs and other pancreatic cystic neoplasms does not deviate from the background signature of negative controls, supporting the concept of a sterile environment. Outlier signals may appear in a small fraction of patients following recent invasive endoscopic procedures. No associations between microbial patterns and clinical or cyst parameters were apparent.

Listeria monocytogenes may survive and persist in food processing environments due to formation of complex multi-species biofilms of environmental microbiota that co-exists in these environments. This study aimed to determine the effect of selected environmental microbiota on biofilm formation and tolerance of L. monocytogenes to benzalkonium chloride in formed biofilms. The studied microbiota included bacterial families previously shown to co-occur with L. monocytogenes in tree fruit packing facilities, including Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae. Biofilm formation ability and the effect of formed biofilms on the tolerance of L. monocytogenes to benzalkonium chloride was measured in single- and multi-family assemblages. Biofilms were grown statically on polystyrene pegs submerged in a R2A broth. Biofilm formation was quantified using a crystal violet assay, spread-plating, confocal laser scanning microscopy, and its composition was assessed using amplicon sequencing. The concentration of L. monocytogenes in biofilms was determined using the most probable number method. Biofilms were exposed to the sanitizer benzalkonium chloride, and the death kinetics of L. monocytogenes were quantified using a most probable number method. A total of 8, 8, 6, and 3 strains of Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae, respectively, were isolated from the environmental microbiota of tree fruit packing facilities and were used in this study. Biofilms formed by Pseudomonadaceae, Xanthomonadaceae, and all multi-family assemblages had significantly higher concentration of bacteria, as well as L. monocytogenes, compared to biofilms formed by L. monocytogenes alone. Furthermore, multi-family assemblage biofilms increased the tolerance of L. monocytogenes to benzalkonium chloride compared to L. monocytogenes mono-species biofilms and planktonic multi-family assemblages. These findings suggest that L. monocytogenes control strategies should focus not only on assessing the efficacy of sanitizers against L. monocytogenes, but also against biofilm-forming microorganisms that reside in the food processing built environment, such as Pseudomonadaceae or Xanthomonadaceae.

We sought to comprehensively profile tissue and cyst fluid in patients with benign, precancerous, and cancerous conditions of the pancreas to characterize the intrinsic pancreatic microbiome.

Small studies in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) have suggested that intra-pancreatic microbial dysbiosis may drive malignant transformation.

Pancreatic samples were collected at the time of resection from 109 patients. Samples included tumor tissue (control, n=20; IPMN, n=20; PDAC, n=19) and pancreatic cyst fluid (IPMN, n=30; SCA, n=10; MCN, n=10). Assessment of bacterial DNA by quantitative PCR and 16S ribosomal RNA gene sequencing was performed. Downstream analyses determined the relative abundances of individual taxa between groups and compared intergroup diversity. Whole-genome sequencing data from 140 patients with PDAC in the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) were analyzed to validate findings.

Sequencing of pancreatic tissue yielded few microbial reads regardless of diagnosis, and analysis of pancreatic tissue showed no difference in the abundance and composition of bacterial taxa between normal pancreas, IPMN, or PDAC groups. Low-grade dysplasia (LGD) and high-grade dysplasia (HGD) IPMN were characterized by low bacterial abundances with no difference in tissue composition and a slight increase in Pseudomonas and Sediminibacterium in HGD cyst fluid. Decontamination analysis using the CPTAC database confirmed a low-biomass, low-diversity intrinsic pancreatic microbiome that did not differ by pathology.

Our analysis of the pancreatic microbiome demonstrated very low intrinsic biomass that is relatively conserved across diverse neoplastic conditions and thus unlikely to drive malignant transformation.

DNA contamination can critically confound microbiome studies. Here, we take a systematic approach to review the current literature and investigate the prevalence of contamination controls in phyllosphere microbiome research over the past decade. By utilising systematic review principles for this review, we were able to conduct a thorough investigation, screening 450 articles from three databases for eligibility and extracting data in a controlled and methodical manner. Worryingly, we observed a surprisingly low usage of both positive and negative contamination controls in phyllosphere research. As a result, we propose a set of minimum standards to combat the effects of contamination in future phyllosphere research.

The early postnatal period represents a critical window of time for the establishment and maturation of the human gut microbiota. The gut microbiota undergoes dramatic developmental changes during the first year of life, being influenced by a variety of external factors, with diet being a major player. Indeed, the introduction of complementary feeding provides novel nutritive substrates and triggers a shift from milk-adapted gut microbiota toward an adult-like bacterial composition, which is characterized by an enhancement in diversity and proportions of fiber-degrading bacterial genera like Ruminococcus, Prevotella, Eubacterium, and Bacteroides genera. Inadequate gut microbiota development in early life is frequently associated with concomitant and future adverse health conditions. Thus, understanding the processes that govern initial colonization and establishment of microbes in the gastrointestinal tract is of great importance. This review summarizes the actual understanding of the assembly and development of the microbial community associated with the infant gut, emphasizing the importance of mother-to-infant vertical transmission events as a fundamental arrival route for the first colonizers.

Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols.

The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood-saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results.

The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood-saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030-0.9998 for SNVs and between 0.8883-0.9991 for small-indels in the case of the WGS protocol, and between 0.8643-0.999 for SNVs and between 0.7781-1.000 for small-indels in the case of the WES protocol.

Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

The microbiome -defined as the microbiota (bacteria, archaea, lower and higher eukaryotes), their genomes, and the surrounding environmental conditions- has a well-described range of physiological functions. Thus, an imbalance of the microbiota composition -dysbiosis- has been associated with pregnancy complications or adverse fetal outcomes. Although there is controversy about the existence or absence of a microbiome in the placenta and fetus during healthy pregnancy, it is known that gut microbiota can produce bioactive metabolites that can enter the maternal circulation and may be actively or passively transferred through the placenta. Furthermore, the evidence suggests that such metabolites have some effect on the fetus. Since the microbiome can influence the epigenome, and modifications of the epigenome could be responsible for fetal programming, it can be experimentally supported that the maternal microbiome and its metabolites could be involved in fetal programming. The developmental origin of health and disease (DOHaD) approach looks to understand how exposure to environmental factors during periods of high plasticity in the early stages of life (e.g., gestational period) influences the program for disease risk in the progeny. Therefore, according to the DOHaD approach, the influence of maternal microbiota in disease development must be explored. Here, we described some of the diseases of adulthood that could be related to alterations in the maternal microbiota. In summary, this review aims to highlight the influence of maternal microbiota on both fetal development and postnatal life, suggesting that dysbiosis on this microbiota could be related to adulthood morbidity.

Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a "kitome", e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.

Sequencing-based approaches for the analysis of microbial communities are susceptible to contamination, which could mask biological signals or generate artifactual ones. Methods for in silico decontamination using controls are routinely used, but do not make optimal use of information shared across samples and cannot handle taxa that only partially originate in contamination or leakage of biological material into controls. Here we present Source tracking for Contamination Removal in microBiomes (SCRuB), a probabilistic in silico decontamination method that incorporates shared information across multiple samples and controls to precisely identify and remove contamination. We validate the accuracy of SCRuB in multiple data-driven simulations and experiments, including induced contamination, and demonstrate that it outperforms state-of-the-art methods by an average of 15-20 times. We showcase the robustness of SCRuB across multiple ecosystems, data types and sequencing depths. Demonstrating its applicability to microbiome research, SCRuB facilitates improved predictions of host phenotypes, most notably the prediction of treatment response in melanoma patients using decontaminated tumor microbiome data.

Reports regarding the presence of bacteria in the fetal environment remain limited and controversial. Recently, extracellular vesicles secreted by the human gut microbiota have emerged as a novel mechanism for host-microbiota interaction. We aimed to investigate the presence of bacterial extracellular vesicles in the fetal environment during healthy pregnancies and determine whether extracellular vesicles derived from the gut microbiota can cross biological barriers to reach the fetus.

Bacterial extracellular vesicles were detectable in the amniotic fluid of healthy pregnant women, exhibiting similarities to extracellular vesicles found in the maternal gut microbiota. In pregnant mice, extracellular vesicles derived from human maternal gut microbiota were found to reach the intra-amniotic space.

Our findings reveal maternal microbiota-derived extracellular vesicles as an interaction mechanism between the maternal microbiota and fetus, potentially playing a pivotal role in priming the prenatal immune system for gut colonization after birth. Video Abstract.

The individual resistance or tolerance against uterine disease in dairy cattle might be related to variations in the uterine tract microbiota. The uterine tract microbiota in dairy cattle is a field of increasing interest. However, its specific taxonomy and functional aspects is under-explored, and information about the microbiota in the endometrium at artificial insemination (AI) is still missing. Although uterine bacteria are likely to be introduced via the vaginal route, it has also been suggested that pathogens can be transferred to the uterus via a hematogenous route. Thus, the microbiota in different layers of the uterine wall may differ. Norwegian Red (NR) is a high fertility breed that also has a high prevalence of subclinical endometritis (SCE), an inflammation of the uterus that has a negative effect on dairy cattle fertility. However, in this breed the negative effect is only moderate, raising the question of whether this may be due to a favorable microbiota. In the present study we investigated the endometrial microbiota in NR at AI by biopsy and cytobrush samples, and comparing this to the vaginal microflora. The second objective was to describe potential differences at both distinct depths of the endometrium, in healthy vs SCE positive NR cows. We sampled 24 lactating and clinically healthy Norwegian red cows in their second heat or more after calving, presented for first AI. First, we obtained a vaginal swab and a cytobrush sample, in addition to a cytotape to investigate the animal's uterine health status with respect to SCE. Secondly, we acquired a biopsy sample from the uterine endometrium. Bacterial DNA from the 16S rRNA gene was extracted and sequenced with Illumina sequencing of the V3-V4 region. Alpha and beta diversity and taxonomic composition was investigated. Our results showed that the microbiota of endometrial biopsies was qualitatively different and more even than that of cytobrush and vaginal swab samples. The cytobrush samples and the vaginal swabs shared a similar taxonomic composition, suggesting that vaginal swabs may suffice to sample the surface-layer uterine microbiota at estrus. The current study gave a description of the microbiota in the healthy and SCE positive NR cows at AI. Our results are valuable as we continue to explore the mechanisms for high fertility in NR, and possible further improvements.

Gestational diabetes mellitus (GDM) is a growing public health concern that affects many pregnancies globally. The condition is associated with adverse maternal and neonatal outcomes including gestational hypertension, preeclampsia, placental abruption, preterm birth, stillbirth, and fetal growth restriction. In the long-term, mothers and children have an increased risk of developing metabolic diseases such as type 2 diabetes and cardiovascular disease. Accumulating evidence suggest that alterations in the maternal microbiome may play a role in the pathogenesis of GDM and adverse pregnancy outcomes. This review describes changes in the maternal microbiome during the physiological adaptations of pregnancy, GDM and adverse maternal and neonatal outcomes. Findings from this review highlight the importance of understanding the link between the maternal microbiome and GDM. Furthermore, new therapeutic approaches to prevent or better manage GDM are discussed. Further research and clinical trials are necessary to fully realize the therapeutic potential of the maternal microbiome and translate these findings into clinical practice.

The placenta is essential for fetal growth and survival and maintaining a successful pregnancy. The sterility of the placenta has been challenged recently; however, the presence of a placental microbiome has been controversial. We tested the hypothesis that the bacterial extracellular vesicles (BEVs) from Gram-negative bacteria as an alternate source of microbial DNA, regardless of the existence of a microbial community in the placenta.

Placentae from the term, not in labor Cesareans deliveries, were used for this study, and placental specimens were sampled randomly from the fetal side. We developed a protocol for the isolation of BEVs from human tissues and this is the first study to isolate the BEVs from human tissue and characterize them.

The median size of BEVs was 130-140 nm, and the mean concentration was 1.8-5.5 × 1010 BEVs/g of the wet placenta. BEVs are spherical and contain LPS and ompA. Western blots further confirmed ompA but not human EVs markers ALIX confirming the purity of preparations. Taxonomic abundance profiles showed BEV sequence reads above the levels of the negative controls (all reagent controls). In contrast, the sequence reads in the same placenta were substantially low, indicating nothing beyond contamination (low biomass). Alpha-diversity showed the number of detected genera was significantly higher in the BEVs than placenta, suggesting BEVs as a likely source of microbial DNA. Beta-diversity further showed significant overlap in the microbiome between BEV and the placenta, confirming that BEVs in the placenta are likely a source of microbial DNA in the placenta. Uptake studies localized BEVs in maternal (decidual) and placental cells (cytotrophoblast), confirming their ability to enter these cells. Lastly, BEVs significantly increased inflammatory cytokine production in THP-1 macrophages in a high-dose group but not in the placental or decidual cells.

We conclude that the BEVs are normal constituents during pregnancy and likely reach the placenta through hematogenous spread from maternal body sites that harbor microbiome. Their presence may result in a low-grade localized inflammation to prime an antigen response in the placenta; however, insufficient to cause a fetal inflammatory response and adverse pregnancy events. This study suggests that BEVs can confound placental microbiome studies, but their low biomass in the placenta is unlikely to have any immunologic impact.