Johnson, Stephen

INTRODUCTION: Intragastric balloons (IGBs) are a safe and effective treatment for obesity. However, limited knowledge exists on the underlying biological changes with IGB placement. METHODS: This single-institution study was part of an adjustable IGB randomized controlled trial. Subjects with obesity were randomized in a 2 is to 1 ratio to 32 weeks of IGB with diet/exercise counseling (n = 8) vs counseling alone (controls, n = 4). Diet/exercise counseling was continued for 24 weeks post-IGB removal to assess weight maintenance. We used mass spectrometry for nontargeted plasma lipidomics analysis and 16S rRNA sequencing to profile the fecal microbiome. RESULTS: Subjects with IGBs lost 15.5% of their body weight at 32 weeks vs 2.59% for controls ( P < 0.05). Maintenance of a 10.5% weight loss occurred post-IGB explant. IGB placement, followed by weight maintenance, led to a −378.9 μM/L reduction in serum free fatty acids compared with pre-IGB (95% confidence interval: 612.9, −145.0). This reduction was mainly in saturated, mono, and omega-6 fatty acids when compared with pre-IGB. Polyunsaturated phosphatidylcholines also increased after IGB placement (difference of 27 μM/L; 95% confidence interval: 1.1, 52.8). Compared with controls, saturated and omega-6 free fatty acids (linoleic and arachidonic acids) were reduced after IGB placement. The fecal microbiota changed post-IGB placement and weight maintenance vs pre-IGB ( P < 0.05). Further analysis showed a possible trend toward reduced Firmicutes and increased Bacteroidetes post-IGB and counseling, a change that was not conclusively different from counseling alone. DISCUSSION: IGB treatment is associated with an altered fecal microbiome profile and may have a better effect on obesity-related lipidome than counseling alone.

Background: Primary sclerosing cholangitis (PSC) is a major risk factor for cholangiocarcinoma (CCA). We investigated biliary and fecal microbiota to determine whether specific microbes in the bile or stool are associated with PSC or CCA. Methods: Bile was obtained from 32 patients with PSC, 23 with CCA with PSC, 26 with CCA without PSC, and 17 controls. Over 90% of bile samples were from patients with perihilar CCA. Stool was obtained from 31 patients with PSC (11 were matched to bile), 16 with CCA with PSC (10 matched to bile), and 11 with CCA without PSC (6 matched to bile). Microbiota composition was assessed using 16SrRNA-marker-based sequencing and was compared between groups. Results: Bile has a unique microbiota distinguished from negative DNA controls and stool. Increased species richness and abundance of Fusobacteria correlated with duration of PSC and characterized the biliary microbiota in CCA. Stool microbiota composition showed no significant differences between groups. Conclusions: We identified a unique microbial signature in the bile of patients with increased duration of PSC or with CCA, suggesting a role for microbiota-driven inflammation in the pathogenesis and or progression to perihilar CCA. Further studies are needed to test this hypothesis.

AbstractAlthough Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection is not uncommon. Here, RNA‐seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. A total of 227 genes with outlier expression were found (164 upregulated and 63 downregulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding β‐hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a resource for identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.

Although Streptococcus agalactiae periprosthetic joint infection (PJI) is not as prevalent as staphylococcal PJI, invasive S. agalactiae infection has recently increased in incidence. Here, RNA-Seq was used to perform transcriptomic analysis of S. agalactiae PJI using fluid derived from sonication of explanted arthroplasties of subjects with S. agalactiae PJI, with results compared to those of S. agalactiae strain NEM316 grown in vitro. 227 genes with outlier expression were found (164 up-regulated and 63 down-regulated) between PJI sonicate fluid and in vitro conditions. Functional enrichment analysis showed genes involved in mobilome and inorganic ion transport and metabolism to be most enriched. Genes involved in nickel, copper, and zinc transport, were upregulated. Among known virulence factors, cyl operon genes, encoding beta-hemolysin/cytolysin, were consistently highly expressed in PJI versus in vitro. The data presented provide insight into S. agalactiae PJI pathogenesis and may be a useful resource for the identification of novel PJI therapeutics or vaccines against invasive S. agalactiae infections.

Abstract The role of maternal microbiome transmitted at birth in cancer control is poorly understood. We have developed the first germfree B6 mouse model of breast cancer to investigate the role of the maternal microbiome in controlling oncogenic/metastatic frequencies of pro-oncogenic mammary cells. In this model, a DNA barcoded, primitive normal mouse mammary epithelial cell encoding MMTV-PyMT oncogene, was transplanted in large numbers into conventional or germfree B6 mice. Next-gen sequencing analysis of the DNA barcodes in tissues enabled us to clonally track millions of cells and measure the frequency and growth dynamics of clones at the primary site and their systemic distribution in circulation and all vital organs, generating an unprecedented high-definition map of cancer progression. Our results show that in conventional B6 mice with maternal microbial transmission at-birth, a small fraction (~0.01%) of transplanted cells transform de novo and produce slow growing, late-onset benign tumors (mouse median survival of &gt; 1 year). In contrast, in germfree B6 mice, a &gt;10-fold higher frequency of cells transform de novo and generate early-onset, highly aggressive metastatic clones, and are frequently associated with features leading to early euthanasia or endpoint (i.e. sudden death, intracardiac metastasis, paralysis, swollen abdomen, early multiorgan aggressive metastasis) (median survival of ~4 months; p&lt;0.005 vs conventional mice). However, postnatal intervention with healthy fecal implants reconstituted gut microbiota in germfree B6 mice and significantly restored protective effects against cancer progression and sudden death, as well as improved quality/duration of life (median survival of &gt;1 year; p&lt;0.005 vs germfree mice; p=ns vs conventional mice). We found maternal microbial transmission fully protected against systemic metastasis. GC-MS analysis identified significantly distinct primary tumor metabolome associated with conventional (non-metastatic) mice vs germfree (metastatic) mice. Metagenomic analysis of fecal samples from a subset of tumor bearing conventional (non-metastatic) mice vs fecal implanted (late-onset, less aggressive metastatic) germfree mice identified significant association between metastasis and altered microbial communities. These findings using novel germfree mouse model of metastasis and cellular DNA barcoding technology provide first direct evidence that diverse microbial communities transmitted at birth play a critical role in protecting the host from clonal de novo mammary cell transformation and progression to systemic disease and thereby improving quality and duration of life. Citation Format: Nagarajan Kannan, Syed Mohammed Musheer Aalam, Xiaojia Tang, Krishna Kalari, Purna Kashyap, Jun Chen, Stephen Johnson, Anguraj Sadanandam, Mark Sherman. Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB226.

Giving birth too soon accounts for half of all newborn deaths worldwide. Clinical symptoms alone are not sufficient to identify women at risk of giving birth too early, as such a pragmatic approach to reducing the incidence of preterm birth entails developing early strategies for intervention before it materializes.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis which is recognized as a major risk factor for cholangiocarcinoma (CCA). We aimed to investigate the roles of the biliary and intestinal microflora in the inflammatory process of PSC and to determine if the microflora is associated with progression to CCA. METHODS: We studied the bile and stool microbiomes in samples obtained from patients with PSC (31 stool and 35 bile samples), sporadic CCA (11 stool and 26 bile samples), and PSC coexisting with CCA (16 stool and bile 17 samples). Control bile was obtained from patients with cholelithiasis or choledocholithiasis (n = 17). DNA extraction was followed by amplification and high-throughput next generation sequencing (MiSeq) of the 16S rDNA (V3-V5) region to identify the microbiota present. We performed: 1. analysis of the stool microbiome between PSC and CCA patients; 2. analysis of the biliary microbiome in PSC and CCA patients; 3. comparison of the biliary microbiome collected before and after biliary stent placement. RESULTS: Although bile samples from study patients had significantly lower sequencing depths than stool, their sequencing depths were much higher than bile from negative controls. The microbiomes of stool and bile samples from the same patients were significantly correlated ( P = 0.022). CCA bile samples had significant difference of alpha diversity from control bile samples with less Streptococcaceae and Desulfovibrionaceae (FDR < 10%) than PSC whereas no significant change of stool microbiome was identified between CCA and PSC. Fusobacterium and Gemella were correlated to PSC duration. An increased abundance of Fusobacteria after stent placement was revealed. Fusobacteria, which is inflammation-promoting, was also increased with the number of stents in the bile duct. CONCLUSION: Our study suggests that the bile microbiome is potentially a more distinctive biomarker than the stool microbiome for PSC and CCA development. There is increased inhabitance of inflammation-promoting bacteria in the bile of CCA patients and after biliary stent placement. Increased PSC duration also increases the microbial burden in the bile, which may also promote CCA development. We further detected specific bile microbes statistically associated with CCA or stent presence in the bile ducts, which raises the possibility of their role in the CCA progression or stent obstruction. Additional analyses with more samples are needed.

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.

Abstract Background Illumina paired-end sequencing has been increasingly popular for 16S rRNA gene-based microbiota profiling. It provides higher phylogenetic resolution than single-end reads due to a longer read length. However, the reverse read (R2) often has significant low base quality, and a large proportion of R2s will be discarded after quality control, resulting in a mixture of paired-end and single-end reads. A typical 16S analysis pipeline usually processes either paired-end or single-end reads but not a mixture. Thus, the quantification accuracy and statistical power will be reduced due to the loss of a large amount of reads. As a result, rare taxa may not be detectable with the paired-end approach, or low taxonomic resolution will result in a single-end approach. Results To have both the higher phylogenetic resolution provided by paired-end reads and the higher sequence coverage by single-end reads, we propose a novel OTU-picking pipeline, hybrid-denovo, that can process a hybrid of single-end and paired-end reads. Using high-quality paired-end reads as a gold standard, we show that hybrid-denovo achieved the highest correlation with the gold standard and performed better than the approaches based on paired-end or single-end reads in terms of quantifying the microbial diversity and taxonomic abundances. By applying our method to a rheumatoid arthritis (RA) data set, we demonstrated that hybrid-denovo captured more microbial diversity and identified more RA-associated taxa than a paired-end or single-end approach. Conclusions Hybrid-denovo utilizes both paired-end and single-end 16S sequencing reads and is recommended for 16S rRNA gene targeted paired-end sequencing data.