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Nance W.C.,University of Michigan | Dowd S.E.,Molecular Research LP MR DNA | Samarian D.,University of Michigan | Chludzinski J.,University of Michigan | And 3 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2013

Few model systems are amenable to developing multi-species biofilms in parallel under environmentallygermane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobialsin the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerousover-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidicsystem that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate theeffectiveness of CPC against oral multi-species biofilms grown in human saliva.Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fedfilter-sterilized saliva for 20 h at 378°C. The bacterial diversity of the biofilms was evaluated by bacterial tagencodedFLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%-0.001% w/v)was examined using Live/Dead stain, CLSM and 3D imaging software.TheanalysisofbiofilmsbybTEFAPdemonstratedthat theycontainedgeneratypically foundinhumandentalplaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella.Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between0.5%and 0.001%w/v. At 0.5%(w/v) CPC, 90%of the total signalwas fromdead/damaged cells. Belowthis concentrationrange, less killing was observed. In the 0.5%-0.05% (w/v) range CPC penetration/killing was greatest andbiofilmthickness was significantly reduced.This work demonstrates the utility of a high-throughput microfluidic-CLSM system to grow multispeciesoral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectivenessof antimicrobials. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. Source


Perkins G.A.,Cornell University | den Bakker H.C.,Cornell University | Burton A.J.,Cornell University | Erb H.N.,Cornell University | And 7 more authors.
Applied and Environmental Microbiology | Year: 2012

Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric disease has not been critically examined. The present study used a combination of 16S rRNA bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescence in situ hybridization (FISH) to characterize the composition and spatial distribution of selected gastric mucosal microbiota of healthy horses. Biopsy specimens of the squamous, glandular, antral, and any ulcerated mucosa were obtained from 6 healthy horses by gastroscopy and from 3 horses immediately postmortem. Pyrosequencing was performed on biopsy specimens from 6 of the horses and yielded 53,920 reads in total, with 631 to 4,345 reads in each region per horse. The microbiome segregated into two distinct clusters comprised of horses that were stabled, fed hay, and sampled at postmortem (cluster 1) and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast (cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse rather than region. The dominant bacteria in cluster 1 were Firmicutes (>83% reads/sample), mainly Streptococcus spp., Lactobacillus spp. and, Sarcina spp. Cluster 2 was more diverse, with predominantly Proteobacteria, Bacteroidetes, and Firmicutes, consisting of Actinobacillus spp. Moraxella spp., Prevotella spp., and Porphyromonas spp. Helicobacter sp. sequences were not identified in any of 53,920 reads. FISH (n=9) revealed bacteria throughout the stomach in close apposition to the mucosa, with significantly more Streptococcus spp. present in the glandular region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota that varies by individual. © 2012, American Society for Microbiology. Source


Cleland E.J.,University of Adelaide | Bassioni A.,University of Adelaide | Boase S.,University of Adelaide | Dowd S.,Molecular Research LP MR DNA | And 2 more authors.
International Forum of Allergy and Rhinology | Year: 2014

Background: Our understanding of fungi in chronic rhinosinusitis (CRS) has been limited by previously employed detection techniques. This study examines the fungal component of the microbiome in CRS patients and controls using a highly sensitive culture-independent molecular technique. The aims of this study include the characterization of fungal richness, prevalence, abundance, temporal changes, and their relationship with patient outcomes. Methods: Swabs were collected from the sinuses of 23 CRS patients and 11 controls. Collection occurred intraoperatively, and at 6 and 12 weeks postoperatively. DNA was extracted from the swabs and fungal outcomes were determined through 18S ribosomal DNA (rDNA) fungal tag-encoded FLX amplicon pyrosequencing. Results: Fungi were ubiquitous to all patients. A total of 207 fungal genera were detected, with a mean sample richness of 8.18 and 12.14 in the control and CRS groups, respectively. Malassezia was detected in all patients at surgery and was also the most abundant. Postoperatively, fungal richness decreased (p < 0.05) and was associated with declines in the prevalence of Fusarium and Neocosmospora (p < 0.05). Neocosmospora was also less abundant postoperatively (p < 0.05). No correlations were found with quality of life. Conclusion: This is the first study to use a highly sensitive pyrosequencing technique to reveal the true diversity of fungi in the sinuses of CRS patients and postoperative changes in richness. The presence of Malassezia, a genus not previously described in the sinuses, is of great interest, and its potential as a disease modifier should see further investigation given its association with atopic disease. © 2014 ARS-AAOA, LLC. Source


Mukherjee N.,University of Memphis | Dowd S.E.,Molecular Research LP MR DNA | Wise A.,WMC TV Action News 5 | Kedia S.,University of Memphis | And 2 more authors.
International Journal of Environmental Research and Public Health | Year: 2014

Public fitness centers and exercise facilities have been implicated as possible sources for transmitting community-acquired bacterial infections. However, the overall diversity of the bacterial community residing on the surfaces in these indoor environments is still unknown. In this study, we investigated the overall bacterial ecology of selected fitness centers in a metropolitan area (Memphis, TN, USA) utilizing culture-independent pyrosequencing of the 16S rRNA genes. Samples were collected from the skin-contact surfaces (e.g., exercise instruments, floor mats, handrails, etc.) within fitness centers. Taxonomical composition revealed the abundance of Firmicutes phyla, followed by Proteobacter and Actinobacteria, with a total of 17 bacterial families and 25 bacterial genera. Most of these bacterial genera are of human and environmental origin (including, air, dust, soil, and water). Additionally, we found the presence of some pathogenic or potential pathogenic bacterial genera including Salmonella, Staphylococcus, Klebsiella, and Micrococcus. Staphylococcus was found to be the most prevalent genus. Presence of viable forms of these pathogens elevates risk of exposure of any susceptible individuals. Several factors (including personal hygiene, surface cleaning and disinfection schedules of the facilities) may be the reasons for the rich bacterial diversity found in this study. The current finding underscores the need to increase public awareness on the importance of personal hygiene and sanitation for public gym users. © 2014 by the authors; licensee MDPI, Basel, Switzerland. Source


Sibley C.D.,University of Calgary | Church D.L.,University of Calgary | Surette M.G.,University of Calgary | Surette M.G.,McMaster University | And 2 more authors.
European Journal of Clinical Microbiology and Infectious Diseases | Year: 2012

The polymicrobial nature of invasive pyogenic infections may be underestimated by routine culture practices, due to the fastidious nature of many organisms and the loss of viability during transport or from prior antibacterials. Pyrosequencing was performed on brain and liver abscesses and pleural fluid and compared to routine culture data. Fortyseven invasive pyogenic infection samples from 44 patients [6 intracerebral abscess (ICA), 21 pyogenic liver abscess (PLA), and 18 pleural fluid (PF) samples] were assayed. Pyrosequencing identified an etiologic microorganism in 100 % of samples versus 45 % by culture, p <0.01. Pyrosequencing was also more likely than traditional cultures to classify infections as polymicrobial, 91 % versus 17 %, p <0.001. The median number of genera identified by pyrosequencing compared to culture was 1 [interquartile range (IQR) 1-3] versus 0 (IQR 0-1) for ICA, 7 (IQR 1- 15) versus 1 (IQR 0-1) for PLA, and 15 (IQR 9-19) versus 0 (IQR 0-1) for PF. Where organisms were cultured, they typically represented the numerically dominant species identified by pyrosequencing. Complex microbial communities are involved in invasive pyogenic infection of the lung, liver, and brain. Defining the polymicrobial nature of invasive pyogenic infections is the first step towards appreciating the clinical and diagnostic implications of these complex communities. © Springer-Verlag 2012. Source

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