Publications by Colleges and Departments (MSU - Bozeman)
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Item Gene expression in pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation(2001-03) Bollinger, Nikki; Hassett, Daniel J.; Iglewski, Barbara H.; Costerton, J. William; McDermott, Timothy R.Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in alasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated thatsodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increasedlasI::lacZ transcription in response to iron limitation, whereas the expression level in the lasmutants was minimal and iron starvation induction oflasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 μM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.Item Resident bacterial flora in the skin of C57BL/6 mice housed under SPF conditions(2010-09) Tavakkol, Zarry; Samuelson, Derrick; Pulcini, Elinor D.; Underwood, Robert A.; Usui, Marcia L.; Costerton, J. William; James, Garth A.; Olerud, John E.; Fleckman, PhilipResearch in cutaneous biology frequently involves models that use mice housed in SPF conditions. Little information is available concerning the species of bacteria that normally inhabit the skin of these mice. The aim of this study was to characterize the bacterial skin flora of mice housed under SPF conditions. Skin biopsies from C57BL/6 mice under normal and surgically prepped conditions were both cultured and analyzed by using DNA extraction and sequencing. The species isolated most commonly from culture were staphylococci. Coagulase-negative staphylococci were isolated more frequently than was Staphylococcus aureus. Molecular sequencing yielded several additional organisms not found by culture. Overall, culturing of isolates yielded 14 species of bacteria, and molecular sequencing identified another 6 species. Investigators conducting cutaneous research in mouse models should aware of the cutaneous bacterial flora present on these mice.Item New methods for the detection of orthopedic and other biofilm infections(2011-03) Costerton, J. William; Post, J. C.; Ehrlich, Garth D.; Hu, Fen Z.; Kreft, R.; Nistico, L.; Kathju, S.; Stoodley, Paul; Hall-Stoodley, Luanne; Maale, G.; James, Garth A.; Sotereanos, N.; DeMeo, P.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.