Browsing by Author "Rhoads, Daniel D."

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Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry
(2017-12) Hamerly, Timothy; Everett, Jake A.; Paris, Nina; Fisher, Steve T.; Karunamurthy, Arivarasan; James, Garth A.; Rumbaugh, Kendra P.; Rhoads, Daniel D.; Bothner, Brian
Monitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue.
Production of cell–cell signaling molecules by bacteria isolated from human chronic wounds
(2010-05) Rickard, A. H.; Colacino, K. R.; Manton, K. M.; Morton, R. I.; Pulcini, Elinor D.; Pfeil, J.; Rhoads, Daniel D.; Wolcott, Randall D.; James, Garth A.
Aim: To (i) identify chronic wound bacteria and to test their ability to produce acyl-homoserine-lactones (AHLs) and autoinducer-2 (AI-2) cellâ€“cell signalling molecules and (ii) determine whether chronic wound debridement samples might contain these molecules. Methods and Results: Partial 16S rRNA gene sequencing revealed the identity of 46 chronic wound strains belonging to nine genera. Using bio-reporter assays, 69-6% of the chronic wound strains were inferred to produce AI-2, while 19-6% were inferred to produce AHL molecules. At least one strain from every genus, except those belonging to the genera Acinetobacter and Pseudomonas, were indicated to produce AI-2. Production of AI-2 in batch cultures was growth-phase dependent. Cross-feeding assays demonstrated that AHLs were produced by Acinetobacter spp., Pseudomonas aeruginosa and Serratia marcescens. Independent from studies of the bacterial species isolated from wounds, AHL and/or AI-2 signalling molecules were detected in 21 of 30 debridement samples of unknown microbial composition. Conclusion: Chronic wound bacteria produce cellâ€“cell signalling molecules. Based on our findings, we hypothesize that resident species generally produce AI-2 molecules, and aggressive transient species associated with chronic wounds typically produce AHLs. Both these classes of cellâ€“cell signals are indicated to be present in human chronic wounds. Significance and Impact of the Study: Interbacterial cellâ€“cell signalling may be an important factor influencing wound development and if this is the case, the presence of AHLs and AI-2 could be used as a predictor of wound severity. Manipulation of cellâ€“cell signalling may provide a novel strategy for improving wound healing.
Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing
(2008-03) Dowd, Scot E.; Sun, Yan; Secor, Patrick R.; Rhoads, Daniel D.; Wolcott, Benjamin M.; James, Garth A.; Wolcott, Randall D.
BACKGROUND:Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate 16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D), venous leg ulcers (V), and pressure ulcers (P). RESULTS:There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods.CONCLUSIONS: If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in order to improve the outcomes of patients. This survey will also foster the pioneering and development of new molecular diagnostic tools, which can be used to identify the community