Citations

Abstract In this review, we aimed to provide insight into the microbiome and its association with endometrial and ovarian cancer and their risk factors. We reviewed the literature focusing on the relationship between the microbiome and cancer, as well as the relationship between gynecologic diseases and cancers. The human body contains different kinds of microorganisms in various body parts, which is termed the microbiome . The number of microorganisms that live in and on the human body is greater than that of the human germ and somatic cells by 10‐fold. The relationship between a human and their microbiome is complex; it is also one of the most important components of homeostasis. Impairment of microbiome–host homeostasis has been associated with obesity, several cancers, preterm labor, inflammatory and allergic conditions and neurodevelopmental disorders. Direct and strong causal relationships have been established for several cancers and microorganisms, such as gastric lymphoma and Helicobacter pylori infection. Interestingly, eradication of the infectious agents has also been shown to be therapeutic. The association between cancer and the microbiome, however, is more complicated than a 1 bacteria–1 cancer model, and a shift in a healthy microbiome can result in various cancers via inflammation, change in microenvironment or DNA‐damaging toxins. The human microbiome is an integral part of homeostasis. Understanding the mechanisms that cause dysbiosis will enable us to elucidate the pathways that result in malignancy and investigate new treatment modalities.

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

The genomic DNA input for stool samples utilized for microbiome composition has not been determined. In this study, we determined the reliable threshold level under which conclusions drawn from the data may be compromised. We also determined the type of microbial bias introduced by less-than-ideal genomic input.

Microbial cells behave differently in colonies and when singled out. The standard methods of microbiome studies present the average characteristics and behaviors of heterogeneous populations and lack the resolution to analyze microbes on a single cell level. Besides, the microbiome does not exist in pure colonies in human bodies, but naturally in complex communities. Therefore, it would be ideal to observe the microbes on a single cell level while maintaining their natural settings. Conventional dilution-to-extinction methods are capable of reducing the complexity of the communities to a minimal ecologically functional unit; however, new tools are needed to perform these studies. Recently, microfluidics as a new technology is gaining attention for various single cell applications because it offers the unique ability of handling nanoscale volume in microstructures, providing an attractive alternative to look at single microbial cells. Here, we compare different microfluidic technologies for microbial single cell studies and review the advancement of microfluidics for various microbial single cell analyses. Continuous microfluidics has been used in microbial single cell culture, whole genome sequencing, gene expression, and metabolic analyses; however, droplet microfluidics is becoming a new trend for its high-throughput single cell encapsulation ability. We envision that different microfluidic paradigms will be integrated in the future for their unique attributes and offer a versatile platform for systematic microbiome studies.

Abstract Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier function and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke create a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) or cancer cases (n=143), we conducted 16S rRNA gene sequencing (MiSeq), with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort. We demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue, indicating a shift in the overall microbial community in lung cancer patients as compared to those without cancer. Lung cancer cases were classified by the relative abundance of two taxa, Variovorax and Streptococcus, with an increase in Variovorax abundance in tumors as compared to non-tumor adjacent paired lung tissue (FDR corrected P=0.013). The species of Variovorax was identified histologically, and also by two additional 16S rRNA strategies (Resphera Insight analysis and PacBio sequencing). A group of taxa were associated with squamous cell carcinoma (SCC), of which Acidovorax were enriched in smokers (P =0.0013). Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD). Therefore, we observed a microbiome-gene and a microbiome-exposure interaction in SCC lung cancer tissue. Together, these results open the door for future biomarker research that could be used to improve screening and direct mechanistic studies of lung cancer therapy. Citation Format: Leigh Greathouse, James White, Valery Bliskovsky, Ashley Vargas, Eric Polley, Elise Bowman, Mohammed Khan, Ana Robles, Brid Ryan, Amiran Dzutsev, Giorgio Trinchieri, Marbin Pineda, Sven Bilke, Paul Meltzer, Marina Walther-Antonio, Garth Ehrlich, Joshua Mell, Joshua Earl, Sergey Balashov, Archana Bhat, Alex Valm, Clayton Deming, Sean Conlan, Julia Oh, Julie Segre, Curtis Harris. Microbiome-TP53 gene interaction in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2017-4925

Abstract Endometrial cancer studies have led to a number of well-defined but mechanistically unconnected genetic and environmental risk factors. One of the emerging modulators between environmental triggers and genetic expression is the microbiome. Because of the inflammatory display in endometrial cancer, we set out to inquire about the composition of the uterine microbiome and its putative role in endometrial cancer. We undertook a pilot study of the microbiome in 238 samples taken from different locations along the entire female reproductive tract in hysterectomy patients with endometrial cancer (n=17), patients with endometrial hyperplasia (n=4) and patients afflicted with a benign uterine condition (n=10). Samples were collected aseptically both in the operating room and the pathology laboratory. Following DNA extraction, the 16S rDNA (V3-V5 region) microbial gene was amplified and sequenced to identify the microbiota present. The microbiome sequencing revealed a structural microbiome shift in the cancer and hyperplasia cases, distinguishable from the benign cases (p=0.01). Several taxa were found to be significantly enriched in samples belonging to the endometrial cancer cohort: Firmicutes (Anaerostipes, ph2, Dialister, Peptoniphilus, 1-68, Ruminococcus, and Anaerotruncus), Spirochaetes (Treponema), Actinobacteria (Atopobium), Bacteroidetes (Bacteroides and Porphyromonas), and Proteobacteria (Arthrospira). Of particular relevance, the simultaneous presence of Atopobium vaginae and an uncultured representative of the Porphyromonas sp. (99% match to P.somerae) were found to be predictive of the disease status (sensitivity 73-93%; specificity 67-90%, AUC 0.918). Sensitivity is increased to 100% if combined with a high vaginal pH (>4.5), although specificity is decreased to 60%. Although our numbers are low, the microbiome signature of hyperplasia was even more distinct from benign disease than endometrial cancer itself. This is suggestive of an early microbiome role in the disease. Given the pathogenic profile of the identified microorganisms these findings raise the possibility of a microbiome role in the etiology or progression of endometrial cancer that should be further investigated. Citation Format: Marina Walther-Antonio, Jun Chen, Francesco Multinu, Alexis Hokenstad, Tammy Distad, Heidi Cheek, Gary Keeney, Douglas Creedon, Heidi Nelson, Andrea Mariani, Nicholas Chia. Endometrial cancer microbiome signature and its implications for carcinogenesis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B45.

Background. Preterm Premature Rupture of Membranes (PPROM) is a major leading cause of preterm births. While the cause for PPROM remains unidentified, it is anticipated to be due to subclinical infection, since a large proportion of PPROM patients display signs of chorioamnionitis. Since subclinical infections can be facilitated by dysbiosis, our goal was to characterize the vaginal microbiome and amniotic fluid discharge upon PPROM, through latency antibiotic treatment, and until delivery, to detect the presence of pathogens, microbiota alteration, and microbial response to treatment. Methods. Enrolled subjects (15) underwent routine institutional antenatal care for PPROM, including the administration of latency antibiotics. Serial vaginal swabs were obtained from diagnosis of PPROM through delivery and the sequencing of the V3–V5 region of the 16S rRNA gene was performed for all collected samples. Results. The results show that Lactobacilli species were markedly decreased when compared to vaginal swabs collected from uncomplicated pregnancy subjects with a matched gestational time. Prevotella and Peptoniphilus were the most prevalent taxa in PPROM subjects at presentation. The vaginal microbiome of the PPROM subjects varied substantially intra- and inter-subjects. Several taxa were found to be significantly reduced during and after the antibiotic treatment: Weeksella , Lachnospira , Achromobacter , and Pediococcus . In contrast, Peptostreptococcus and Tissierellaceae ph2 displayed a significant increase after the antibiotic treatment. However, the relative abundance of Lactobacillus , Prevotella , and Peptoniphilus was not substantially impacted during the hospitalization of the PPROM subjects. The deficiency of Lactobacillus , and constancy of known pathogenic species, such as Prevotella and Peptoniphilus during and after antibiotics, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches. Discussion. PPROM is responsible for one third of all preterm births. It is thought that subclinical infection is a crucial factor in the pathophysiology of PPROM because 25–40% of patients present signs of chorioamnionitis on amniocentesis. Here we sought to directly assess the bacterial content of the vagina and leaking amniotic fluid of subjects at presentation, throughout treatment and up until delivery, in order to search for common pathogens, microbiota changes, and microbial response to latency antibiotic treatment. We have found that the vaginal microbiome of PPROM subjects is highly variable and displays significant changes to treatment. However, the unchanging deficiency of Lactobacillus , and persistence of known pathogenic species, such as Prevotella and Peptoniphilus from presentation, through antibiotic treatment and up until delivery, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches.

The presence of microbialite structures in a freshwater, dimictic mid-latitudelake and their establishment after the last ice age about 10,000 years ago is puzzling.Freshwater calcite microbialites at Pavilion Lake, British Columbia, Canada, consist of acomplex community of microorganisms that collectively form large, ordered structuredaggregates. This distinctive assemblage of freshwater calcite microbialites was studied through standard microbial methods, morphological observations, phospholipid fatty acid(PLFA) analysis, DNA sequencing and the identification of quorum sensing molecules.Our results suggest that the microbialites may represent a transitional form from theexclusively prokaryotic colonial precursors of stromatolites to the multicellular organismicaggregates that give rise to coral reefs.