Kate Bowie

Wastewater surveillance (WS) has been widely adopted as a cost-effective and population-representative infectious disease monitoring tool and is increasingly being applied to bacterial and antimicrobial resistance gene (ARG) targets. However, some of these targets may persist in pipe biofilms and detach into wastewater, complicating accurate WS interpretation. To investigate biofilm contributions to wastewater pathogen and ARG signals, paired sink-drain biofilm, branch-drain-plumbing biofilm (“sewer biofilm”), and wastewater were collected from five hospital sites over a four-month period and analyzed using 16S rRNA gene amplicon sequencing and probe-capture metagenomics. Overall, sewer biofilm bacterial communities were as diverse as wastewater. Across sites, a mean of 9% (0.9–23.3%) of wastewater bacterial communities could be attributed to sewer biofilm communities. Many clinically relevant pathogens were consistently detected both in sewer biofilm and wastewater, including environmentally persistent and/or biofilm-associated taxa (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae) . While many ARGs overlapped between wastewater and biofilms (e.g., tetA, sul1, blaCTX-M, vanA ), others were significantly enriched in sewer biofilms (e.g., qacL, van -operon and OXA genes). Together, these findings confirm that wastewater pathogen and resistome profiles integrate inputs from both human shedding and pipe-resident communities and therefore need to be considered when selecting WS targets and interpreting signal. Synopsis: Wastewater surveillance signal reflects bacteria from both human shedding and sewer-resident microbiomes. Paired biofilm and wastewater analyses demonstrated overlapping pathogens and resistomes, indicating pipe biofilms can shape signals used for population-level surveillance. TOC

Antimicrobial resistant pathogens and associated infections represent major public health threats affecting healthcare facilities, with sink drain biofilms serving as reservoirs for many of these bacteria. Despite attempts at sink drain biofilm disinfection and removal, drain biofilms inevitably regrow, and disinfection may shape the returning microbial communities and their resistance profiles. We applied culture-based and metagenomic approaches to study these drain disinfection effects on microbial community abundance, taxonomy, and antimicrobial resistance in operational hospital sinks. Drain biofilms regrew to baseline densities in approximately four days. Regrown biofilms contained more viable carbapenem-resistant bacteria and were dominated by Pseudomonadota, including Cupriavidus and Pseudomonas. Long-read sequencing revealed an increase in multidrug efflux pump genes after disinfection, which confer broad resistance to antibiotics and disinfectants. This work provides mechanistic insights into how disinfection influences sink drain biofilm ecology and the enrichment of antimicrobial resistance, with implications for infection prevention strategies in healthcare environments.

Antimicrobial resistant pathogens and associated infections represent major public health threats affecting healthcare facilities, with sink drain biofilms serving as reservoirs for many of these bacteria. Despite attempts at sink drain biofilm disinfection and removal, drain biofilms inevitably regrow, and disinfection may shape the returning microbial communities and their resistance profiles. We applied culture-based and metagenomic approaches to study these drain disinfection effects on microbial community abundance, taxonomy, and antimicrobial resistance in operational hospital sinks. Drain biofilms regrew to baseline densities in approximately four days. Regrown biofilms contained more viable carbapenem-resistant bacteria and were dominated by Pseudomonadota, including Cupriavidus and Pseudomonas . Long-read sequencing revealed an increase in multidrug efflux pump genes after disinfection, which confer broad resistance to antibiotics and disinfectants. This work provides mechanistic insights into how disinfection influences sink drain biofilm ecology and the enrichment of antimicrobial resistance, with implications for infection prevention strategies in healthcare environments.

Background Several studies have identified bacteria and other microbes in the bladder and lower urinary tract in the absence of infection. In women, the urinary microbiome has been associated with lower urinary tract symptoms (LUTS), however, similar studies have not been undertaken in large cohorts of men. Here we examine the urinary microbiome and its association with LUTS in a subset of 500 men aged 65–90 years from the Osteoporotic Fractures in Men (MrOS) study. Methods Bacterial DNA was isolated from urine samples. The V4 region of the 16S rRNA gene was sequenced using Illumina Miseq. Microbiome characteristics, including diversity measures and urotypes, are examined for associations with clinical characteristics and lower urinary tract symptoms. Results Here we identify significant associations between benign prostatic hyperplasia (BPH), age, and body mass index (BMI) with several diversity metrics. Our analysis reveals complex relationships between BMI, BPH, LUTS, and alpha diversity which give insight into the intricate dynamics of the urinary microbiome. Conclusions By beginning to uncover the interrelationships of BPH, BMI, LUTS, and the urinary microbiome, these results can inform future study design to better understand the heterogeneity of the male urinary microbiome.

In the absence of infection, blood has previously been understood to be free of microbes. However, with advances in sequencing technology this notion has been challenged, prompting new investigations into microbial DNA within the blood of both healthy and diseased individuals. To comprehensively survey microbial DNA in blood, we separated blood into fractions (plasma, red blood cells, and buffy coat) and assessed if the microbial-DNA is cell-free by the addition of DNase to a subset of each fraction. We measured 16S rRNA gene copy number with digital droplet PCR and identified the taxonomic origin of the microbial DNA with synthetic full-length 16S rRNA gene sequencing. As a use case, we examine microbial DNA from the blood of 5 men without prostate cancer (PC), 5 men with low-grade PC, and 5 men with high-grade PC. Our study demonstrates that the majority of microbial DNA is cell-free, indicating that it is not representative of proliferating microbes. Our analyses also revealed buffy coat had the lowest number of 16S rRNA gene copies yet highest number of genera of the fractions (median 23.3 copies/µL and 10 genera) and thus may be a useful fraction to study moving forward. Additionally, microbial DNA in blood may have utility as a biomarker, as we detected disease-associated compositional differences in the plasma and buffy coat fractions. This study lays the groundwork for rigorously studying microbial DNA in blood, however larger studies are needed to confirm our disease-association findings. Importance The concept of a “blood microbiome” has sparked debate in recent years, with questions about whether microbes truly exist in circulation. This study provides a crucial evaluation of the fractions of blood and their capacity to harbor microbial DNA, offering important context for prior and future research. By using DNase to differentiate between cell-associated and cell-free microbial DNA, we show that while microbial DNA is present in blood, it is sparse and heterogenous. These findings highlight the need for rigorous study design that carefully considers both positive and negative controls, as well as the specific blood fractions being examined.

Several studies have identified bacteria and other microbes in the bladder and lower urinary tract in the absence of infection. In women, the urinary microbiome has been associated with lower urinary tract symptoms (LUTS), however, similar studies have not been undertaken in large cohorts of men. Here we examine the urinary microbiome and its association with LUTS in a subset of 500 men aged 65 to 90 years from the Osteoporotic Fractures in Men (MrOS) study. We identified significant associations between benign prostatic hyperplasia (BPH), age, and body mass index (BMI) with several diversity metrics. Our analysis revealed complex relationships between BMI, BPH, LUTS, and alpha diversity which give insight into the intricate dynamics of the urinary microbiome. By beginning to uncover the interrelationships of BPH, BMI, LUTS, and the urinary microbiome, these results can inform future study design to better understand the heterogeneity of the male urinary microbiome.

Non-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients. In vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers. We found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth. These studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression.

Purpose: Although high glucocorticoid receptor (GR) expression in early-stage estrogen receptor (ER)-negative breast cancer is associated with shortened relapse-free survival (RFS), how associated GR transcriptional activity contributes to aggressive breast cancer behavior is not well understood. Using potent GR antagonists and primary tumor gene expression data, we sought to identify a tumor-relevant gene signature based on GR activity that would be more predictive than GR expression alone. Experimental Design: Global gene expression and GR ChIP-sequencing were performed to identify GR-regulated genes inhibited by two chemically distinct GR antagonists, mifepristone and CORT108297. Differentially expressed genes from MDA-MB-231 cells were cross-evaluated with significantly expressed genes in GR-high versus GR-low ER-negative primary breast cancers. The resulting subset of GR-targeted genes was analyzed in two independent ER-negative breast cancer cohorts to derive and then validate the GR activity signature (GRsig). Results: Gene expression pathway analysis of glucocorticoid-regulated genes (inhibited by GR antagonism) revealed cell survival and invasion functions. GR ChIP-seq analysis demonstrated that GR antagonists decreased GR chromatin association for a subset of genes. A GRsig that comprised n = 74 GR activation-associated genes (also reversed by GR antagonists) was derived from an adjuvant chemotherapy-treated Discovery cohort and found to predict probability of relapse in a separate Validation cohort (HR = 1.9; P = 0.012). Conclusions: The GRsig discovered herein identifies high-risk ER-negative/GR-positive breast cancers most likely to relapse despite administration of adjuvant chemotherapy. Because GR antagonism can reverse expression of these genes, we propose that addition of a GR antagonist to chemotherapy may improve outcome for these high-risk patients. Clin Cancer Res; 24(14); 3433–46. ©2018 AACR.

In estrogen receptor (ER)–negative breast cancer, high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER⁺ relapse-free survival (RFS; independently of progesterone receptor expression). To understand the mechanism by which GR expression is associated with a better ER⁺ breast cancer outcome, the global effect of GR-mediated transcriptional activation in ER⁺ breast cancer cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing in ER⁺/GR⁺ MCF-7 cells revealed that upon coactivation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased GR chromatin association was observed at ER, FOXO, and AP1 response elements. In addition, ER associated with GR response elements, suggesting that ER and GR interact in a complex. Coactivation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g., KDM4B, VDR) and inhibiting the Wnt signaling pathway (IGFBP4). Finally, expression of these individual prodifferentiation genes was associated with significantly improved RFS in ER⁺ breast cancer patients. Together, these data suggest that the coexpression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage breast cancer by coordinating the altered expression of genes favoring differentiation. Implications: The interaction between ER and GR activity highlights the importance of context-dependent nuclear receptor function in cancer. Mol Cancer Res; 14(8); 707–19. ©2016 AACR.