In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia we chose to investigate the ET-743 (Yondelis®) biosynthetic pathway. This molecule is an approved anti-cancer agent obtained in low abundance (10−4–10−5% w/w) from the tunicate Ecteinascidia turbinata, and is generated in suitable quantities for clinical use by a lengthy semi-synthetic process. Based on structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anti-cancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogs through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P ‫؍‬ 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Javad, "Successful identification of pathogens by polymerase chain reaction (PCR)-based electron spray ionization time-of-flight mass spectrometry (ESI-TOF-MS) in culture-negative periprosthetic joint infection." (2012). Rothman Institute. Paper 22.

The detection and identification of bacteria present in natural and industrial ecosystems is now entirely based on molecular systems that detect microbial RNA or DNA. Culture methods were abandoned, in the 1980s, because direct observations showed that <1% of the bacteria in these systems grew on laboratory media. Culture methods comprise the backbone of the Food and Drug Administration-approved diagnostic systems used in hospital laboratories, with some molecular methods being approved for the detection of specific pathogens that are difficult to grow in vitro. In several medical specialties, the reaction to negative cultures in cases in which overt signs of infection clearly exist has produced a spreading skepticism concerning the sensitivity and accuracy of traditional culture methods. We summarize evidence from the field of orthopedic surgery, and from other medical specialties, that support the contention that culture techniques are especially insensitive and inaccurate in the detection of chronic biofilm infections. We examine the plethora of molecular techniques that could replace cultures in the diagnosis of bacterial diseases, and we identify the new Ibis technique that is based on base ratios (not base sequences), as the molecular system most likely to fulfill the requirements of routine diagnosis in orthopedic surgery.

Bacterial biofilms have been observed in many prosthesis-related infections, and this mode of growth renders the infection both difficult to treat and especially difficult to detect and diagnose using standard culture methods. We (1) tested a novel coupled PCR-mass spectrometric (PCR-MS) assay (the Ibis T5000) on an ankle arthroplasty that was culture negative on preoperative aspiration and then (2) confirmed that the Ibis assay had in fact detected a viable multispecies biofilm by further micrographic and molecular examinations, including confocal microscopy using Live/Dead stain, bacterial FISH, and reverse-transcriptase-PCR (RT-PCR) assay for bacterial mRNA. The Ibis technology detected Staphylococcus aureus, Staphylococcus epidermidis, and the methicillin resistance gene mecA in soft tissues associated with the explanted hardware. Viable S. aureus were confirmed using RT-PCR, and viable cocci in the biofilm configuration were detected microscopically on both tissue and hardware. Species-specific bacterial FISH confirmed a polymicrobial biofilm containing S. aureus. A novel culture method recovered S. aureus and S. epidermidis (both methicillin resistant) from the tibial metal component. These observations suggest that molecular methods, particularly the new Ibis methodology, may be a useful adjunct to routine cultures in the detection of biofilm bacteria in prosthetic joint infection.

BackgroundToll-like receptors (TLRs) recognize known molecules from microbes and have an established role in tumorigenesis. Using a rat model of esophageal adenocarcinoma, and human clinical samples, we investigated genes central to TLR-mediated signal transduction and characterized the esophageal microbiome across the spectrum of esophageal adenocarcinoma carcinogenesis.MethodsWe surgically induced bile/acid reflux in rats and their esophagi were harvested at 40 weeks post-surgery. Tissue samples from the model were selected for gene expression profiling. Additionally, for rat and human samples microbiome analysis was performed using PCR-ESI-MS-TOF technology with validation by fluorescence in situ hybridization.ResultsGene expression results in the rat model indicated a significant upregulation of TLRs 1-3, 6, 7 and 9 in EAC compared to normal epithelium. PCR-ESI-MS-TOF analysis revealed a prevalence of Escherichia coli in Barrett’s esophagus (60 %) and esophageal adenocarcinoma (100 %), which was validated by fluorescence in situ hybridization. In the human clinical samples, Streptococcus pneumonia was detected in high abundance in gastroesophageal reflux disease and Barrett’s esophagus (50–70 %) in comparison to tumor adjacent normal epithelium, dysplasia, and esophageal adenocarcinoma (20–30 %). E. coli was detected in the Barrett’s esophagus and esophageal adenocarcinoma groups but was absent in the tumor adjacent normal epithelium, dysplasia, and the gastroesophageal reflux disease groups.ConclusionsWe demonstrated an association between the TLR signaling pathway and E. coli hinting towards possible early molecular changes being mediated by microbes in the rat model of esophageal adenocarcinoma carcinogenesis. Studies on human clinical samples also corroborate results to some extent; however, a study with larger sample size is needed to further explore this association.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2093-8) contains supplementary material, which is available to authorized users.

Microbial infections delay wound healing, but the effect of the composition of the wound microbiome on healing parameters is unknown. To better understand bacterial communities in chronic wounds, we analyzed debridement samples from lower-extremity venous insufficiency ulcers using the following: conventional anaerobic and aerobic bacterial cultures; the Ibis T5000 universal biosensor (Abbott Molecular); and 16S 454 FLX titanium series pyrosequencing (Roche). Wound debridement samples were obtained from 10 patients monitored clinically for at least 6 months, at which point 5 of the 10 sampled wounds had healed. Pyrosequencing data revealed significantly higher bacterial abundance and diversity in wounds that had not healed at 6 months. Additionally, Actinomycetales was increased in wounds that had not healed, and Pseudomonadaceae was increased in wounds that had healed by the 6-month follow-up. Baseline wound surface area, duration, or analysis by Ibis or conventional culture did not reveal significant differences between wounds that healed after 6 months and those that did not. Thus, pyrosequencing identified distinctive baseline characteristics of wounds that did not heal by the 6-month follow-up, furthering our understanding of potentially unique microbiome characteristics of chronic wounds.

Introduction We used next-generation, state-of-the-art, culture-independent methodology to survey urine microbiota of UCPPS males and control participants enrolled in the MAPP Network to investigate a possible microbial etiology. Methods Male UCPPS patients and matched controls were asked to provide VB1, VB2 and VB3 urine specimens. Specimens were analyzed with Ibis T-5000 Universal Biosensor technology to provide comprehensive identification of bacterial and select fungal species. Differences between UCPPS and control study participants for presence of species or species variation within a higher taxonomic grouping (genus) were evaluated using permutational multivariate analysis of variance and logistic regression. Results VB1 and VB2 urine specimens were obtained from 110 (VB3 in 67) UCPPS participants and 115 (VB3 in 62) controls. A total of 78, 73 and 54 species (42, 39 and 27 genera) were detected in VB1, VB2 and VB3 respectively. Mean (SD) VB1, VB2 and VB3 species count per person was 1.62 (1.28), 1.38 (1.36) and 1.33(1.24) for cases and 1.75(1.32), 1.23(1.15) and 1.56 (0.97) for controls respectively. Overall species and genus composition differed significantly between UCPPS and control participants in VB1 (p=0.002 species level, p=0.004 genus level) with Burkholderia cenocepacia over represented in UCPPS cases. No significant differences were observed at any level in VB2 or VB3 samples. Conclusions Assessment of baseline culture-independent microbiological data from male subjects enrolled in the MAPP Network has identified over representation of B cenocepacia in UCPPS. Future studies are planned to further evaluate microbiota associations with variable and changing UCPPS symptom patterns.