The Pseudomonas aeruginosa biofilm matrix and cells are drastically impacted by gas discharge plasma treatment: A comprehensive model explaining plasma-mediated biofilm eradication.

Biofilms are microbial communities encased in a protective matrix composed of exopolymeric substances including exopolysaccharides, proteins, lipids, and extracellular DNA. Biofilms cause undesirable effects such as biofouling, equipment damage, prostheses colonization, and disease. Biofilms are also more resilient than free-living cells to regular decontamination methods and therefore, alternative methods are needed to eradicate them. The use of non-thermal atmospheric pressure plasmas is a good alternative as plasmas contain reactive species, free radicals, and UV photons well-known for their decontamination potential against free microorganisms. Pseudomonas aeruginosa biofilms colonize catheters, indwelling devices, and prostheses. Plasma effects on cell viability have been previously documented for P. aeruginosa biofilms. Nonetheless, the effect of plasma on the biofilm matrix has received less attention and there is little evidence regarding the changes the matrix undergoes. The aim of this work was to study the effect plasma exerts mostly on the P. aeruginosa biofilm matrix and to expand the existing knowledge about its effect on sessile cells in order to achieve a better understanding of the mechanism/s underlying plasma-mediated biofilm inactivation. We report a reduction in the amount of the biofilm matrix, the loss of its tridimensional structure, and morphological changes in sessile cells at long exposure times. We show chemical and structural changes on the biofilm matrix (mostly on carbohydrates and eDNA) and cells (mostly on proteins and lipids) that are more profound with longer plasma exposure times. We also demonstrate the presence of lipid oxidation products confirming cell membrane lipid peroxidation as plasma exposure time increases. To our knowledge this is the first report providing detailed evidence of the variety of chemical and structural changes that occur mostly on the biofilm matrix and sessile cells as a consequence of the plasma treatment. Based on our results, we propose a comprehensive model explaining plasma-mediated biofilm inactivation.

Biodiversity of cultivable Burkholderia species in Argentinean soils under no-till agricultural practices.

No-tillage crop production has revolutionized the agriculture worldwide. In our country more than 30 Mha are currently cultivated under no-till schemes, stressing the importance of this management system for crop production. It is widely recognized that soil microbiota is altered under different soil managements. In this regard the structure of Burkholderia populations is affected by soils management practices such as tillage, fertilization, or crop rotation. The stability of these structures, however, has not been evaluated under sustainable schemes where the impact of land practices could be less deleterious to physicochemical soils characteristics. In order to assess the structure of Burkholderia spp. populations in no-till schemes, culturable Burkholderia spp. strains were quantified and their biodiversity evaluated. Results showed that Burkholderia spp. biodiversity, but not their abundance, clearly displayed a dependence on agricultural managements. We also showed that biodiversity was mainly influenced by two soil factors: Total Organic Carbon and Total Nitrogen. Results showed that no-till schemes are not per se sufficient to maintain a richer Burkholderia spp. soil microbiota, and additional traits should be considered when sustainability of productive soils is a goal to fulfil productive agricultural schemes.

Plasma-mediated inactivation of Pseudomonas aeruginosa biofilms grown on borosilicate surfaces under continuous culture system.

Biofilms are microbial communities attached to a surface and embedded in a matrix composed of exopolysaccharides and excreted nucleic acids. Bacterial biofilms are responsible for undesirable effects such as disease, prostheses colonization, biofouling, equipment damage, and pipe plugging. Biofilms are also more resilient than free-living cells to regular sterilization methods and therefore it is indispensable to develop better ways to control and remove them. The use of gas discharge plasmas is a good alternative since plasmas contain a mixture of reactive agents well-known for their decontamination potential against free microorganisms. We have previously reported that Pseudomonas aeruginosa biofilms were inactivated after a 1-min plasma exposure. We determined that the adhesiveness and the thickness of Pseudomonas biofilms grown on borosilicate were reduced. We also reported sequential morphological changes and loss of viability upon plasma treatment. However, the studies were carried out in batch cultures. The use of a continuous culture results in a more homogenous environment ensuring reproducible biofilm growth. The aim of this work was to study plasma-mediated inactivation of P. aeruginosa biofilms grown on borosilicate in a continuous culture system. In this paper we show that biofilms grown on glass under continuous culture can be inactivated by using gas discharge plasma. Both biofilm architecture and cell culturability are impacted by the plasma treatment. The inactivation kinetics is similar to previously described ones and cells go through sequential changes ranging from minimal modification without loss of viability at short plasma exposure times, to major structure and viability loss at longer exposure times. We report that changes in biofilm structure leading to the loss of culturability and viability are related to a decrease of the biofilm matrix adhesiveness. To our knowledge, there has been no attempt to evaluate the inactivation/sterilization of biofilms grown in a continuous system.

Cal Poly Pomona NUE Project: Implementing Microscale and Nanoscale Investigations Throughout the Undergraduate Curriculum.

NUE funded work at California State Polytechnic University involved development and implementation of nanotechnology modules for physics courses spanning all levels of the undergraduate curriculum, from freshman service courses to senior level laboratories and independent research projects. These modules demonstrate the application of fundamental physics at the nanoscale that complement macroscopic investigations. The introductory level and some of the advanced level modules have been described previously in journal papers and will be outlined briefly here. The main focus of this article, however, is to describe some newer work involving nanoscale experiments that have been developed for senior level laboratories and independent research. These experiments involve applications as diverse as tunneling diodes, gas discharge plasmas for biofilm inactivation, and quantized conductance in gold nanowires.

Pseudomonas aeruginosa Biofilm Inactivation: Decreased Cell Culturability, Adhesiveness to Surfaces, and Biofilm Thickness Upon High-Pressure Nonthermal Plasma Treatment.

Bacterial biofilms are more resilient to standard killing methods than free-living bacteria. Pseudomonas aeruginosa PAO1 biofilms grown on borosilicate coupons were treated with gas-discharge plasma for various exposure times. Almost 100% of the cells were inactivated after a 5-min plasma exposure. Atomic force microscopy was used to image the biofilms and study their micromechanical properties. Results show that the adhesiveness to borosilicate and the thickness of the Pseudomonas biofilms are reduced upon plasma treatment.

Is gas-discharge plasma a new solution to the old problem of biofilm inactivation?

Conventional disinfection and sterilization methods are often ineffective with biofilms, which are ubiquitous, hard-to-destroy microbial communities embedded in a matrix mostly composed of exopolysaccharides. The use of gas-discharge plasmas represents an alternative method, since plasmas contain a mixture of charged particles, chemically reactive species and UV radiation, whose decontamination potential for free-living, planktonic micro-organisms is well established. In this study, biofilms were produced using Chromobacterium violaceum, a Gram-negative bacterium present in soil and water and used in this study as a model organism. Biofilms were subjected to an atmospheric pressure plasma jet for different exposure times. Our results show that 99.6 % of culturable cells are inactivated after a 5 min treatment. The survivor curve shows double-slope kinetics with a rapid initial decline in c.f.u. ml(-1) followed by a much slower decline with D values that are longer than those for the inactivation of planktonic organisms, suggesting a more complex inactivation mechanism for biofilms. DNA and ATP determinations together with atomic force microscopy and fluorescence microscopy show that non-culturable cells are still alive after short plasma exposure times. These results indicate the potential of plasma for biofilm inactivation and suggest that cells go through a sequential set of physiological and morphological changes before inactivation.

Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici.

Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features. The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules. Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici. This work shows that R. gallicum and R. tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia. Moreover, some of these host plants can be nodulated by both species. The results confirm the great promiscuity of R. tropici and also support the hypothesis that the species R. gallicum may be native from America or cosmopolitan and worldwide spread.

Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti.

Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.

Nitrogen limitation of chemostat-grown Rhizobium etli elicits higher infection-thread formation in Phaseolus vulgaris.

The symbiotic association between rhizobia and legume roots is a complex process involving many steps. An infection thread is a tubular structure of host origin formed during the infection of legume roots by rhizobia. Previous studies with batch cultures have reported that optimal attachment of rhizobia to root hairs coincides with nutrient limitation. In this study, the ability of chemostat-grown, nutrient-limited Rhizobium etli cells to form infection threads with its symbiotic partner Phaseolus vulgaris was investigated. Rhizobia were grown in a chemostat in synthetic media under C- or N-limiting conditions. Infection-thread formation was examined after inoculation of seedlings with a rhizobial cell suspension from each treatment. The number of infection threads was estimated by light microscopy after staining root sections with o-toluidine. Exopolysaccharide (EPS) production was also measured, and the cellular content and electrophoretic pattern of lipopolysaccharide (LPS) determined semiquantitatively. N-limited cells showed a markedly higher infectivity (measured as infection-thread formation) than C-limited cells. With one of the two bean cultivars used, the number of infection threads produced by N-limited cells was higher than that produced by exponentially growing cells in batch cultures. The higher infectivity of N-limited cells was correlated with higher EPS production. Electrophoretic analysis of LPS showed that C- and N-limited cells shared a common profile but the relative concentration of short LPS forms differed.