Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 10²–10⁵ colony forming units g⁻¹ fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR^+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 10⁵ – 4.0 × 10⁷ colony forming units g⁻¹ tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.     

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of moderately halophilic bacteria isolated from the Weihai Solar Saltern (China)

A total of 45 moderately halophilic bacteria was isolated from sediment and saline water collected from the Weihai Solar Saltern (China). The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The halophilic strains were tested for their antimicrobial activity. Cytotoxicity assay was performed to determine which of the halophilic strains could inhibit proliferation of human hepatocellular carcinoma Bel 7402 cells. Our results showed that all of the isolated 45 strains displayed moderately halophilic characteristics. Phylogenetic analysis indicated that 17 of the isolated strains were related to the phylum Firmicutes and belonged to four genera, Bacillus, Halobacillus, Planococcus and Salinicoccus. The other strains identified as genus of Halomonas belonged to phylum γ-Proteobacteria. Most of the halophilic bacterial strains showed potent activities against Gram-positive bacteria, human pathogenic fungi and plant pathogenic fungi. In addition, the crude extracts from 14 halophilic bacterial strains showed cytotoxic activity against tumor cells Bel 7402, and five of them showed remarkable activities with IC₅₀ less than 40 μg ml⁻¹. Our results suggest that the moderately halophilic bacteria may be developed as promising sources for the discovery of novel bioactive substances.     

Mycotoxins of Alternaria alternata produced on ceiling tiles

The production of mycotoxins by Alternaria alternata in cellulosic ceiling tiles was examined with thin-layer chromatography and high-performance liquid chromatography procedures. Alternariol and alternariol monomethyl ether were found in ceiling tile extracts, whereas extracts of control rice cultures of all three isolates produced these mycotoxins plus altenuene and altertoxin I. Extensive fungal growth and mycotoxin production occurred in the ceiling tiles at relative humidities of 84–89% and 97%. Received 28 May 1997/ Accepted in revised form 06 October 1997     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.     

Recovery of plant growth-promoting rhizobacteria from sodium alginate beads after 3 years following storage at 4 °C

Two plant growth-promoting bacteria, Bacillus subtilis and Pseudomonas corrugata, immobilized in a sodium alginate based formulation were evaluated for their survival, viability and plant growth-promoting ability after 3 years of storage at 4 °C. Populations of both of the bacterial isolates recovered from the immobilized sodium alginate beads were in the order of 10⁸ cfu g⁻¹. The plant-based bioassay indicated that the plant growth promotion ability of both of the bacterial isolates was equal to those of fresh broth-based formulations. The bacterial isolates retained the root colonization, and antifungal and enzyme activities in the alginate-based formulation during storage.     

Airborne bacteria, fungi, and endotoxin levels in residential microenvironments: a case study

Limited data are currently available on the concentrations of airborne bacteria, fungi, and endotoxins in indoor environments. The levels of aerial bacteria and fungi were measured at several microenvironments within a well-ventilated residential apartment in Singapore including the living room, kitchen, bedroom, toilet, and at a workplace environment by sampling indoor air onto culture medium plates using the 6-stage Andersen sampler. Total microbial counts were determined by collecting the air samples in water with the Andersen sampler, staining the resultant extracts with a fluorescent dye, acridine orange, and counting the microbes using a fluorescent microscope. The levels of airborne endotoxins were also determined by sampling the airborne microorganisms onto 0.4?μm polycarbonate membrane filter using the MiniVol sampler at 5?l/min for 20?h with a PM_2.5 cut-off device. The aerial bacterial and fungal concentrations were found to be in the ranges of 117–2,873?CFU/m³ and 160–1,897?CFU/m³, respectively. The total microbial levels ranged from 49,000 to 218,000?microbes/m³. The predominant fungi occurring in the apartment were Aspergillus and Penicillium while the predominant bacterial strains appeared to be Staphylococcus and Micrococcus. The average indoor endotoxin level was detectable in the range of 6–39?EU/m³. The amount of ventilation and the types of human activities carried out in the indoor environment appeared to be important factors affecting the level of these airborne biological contaminants.     

A root tissue culture system to study winter wheat-rhizobacteria interactions

Summary A tissue culture procedure was developed for growing winter wheat roots. This system was used to study bacterial-root interactions and root colonization by Pseudomonas cepacia strains R55 and R85, Azospirillum brasilense ATCC 29729 and Azotobacter chroococcum ATCC 9043. Axenic root tissue cultures were inoculated with bacteria and incubated at 25° C on a rotary shaker (150 rpm) for up to 3 weeks. At various intervals, root morphology and root hair development, bacterial colonization of root surfaces and nitrogenase activity were determined. As determined by plate counting techniques, bacterial attachment to the roots varied from 7.5 × 10⁴ to 3.2 × 10⁷ colony-forming units cm^–1. Scanning electron microscopy of inoculated roots revealed that some rhizobacteria (e.g., P. cepacia R85) significantly enhanced root hair development, and others (e.g., P. cepacia R55) specifically colonized root exudation sites. Nitrogenase activity of roots inoculated with either A. brasilense ATCC 29729 or A. chroococcum ATCC 9043 was stimulated if the inorganic nitrogen sources in the medium were replaced with 300 g glutamine ml^–1. We propose that the use of root tissue culture systems will facilitate studies on plant root-microbe interactions.Contribution no. R 626, Saskatchewan Institute of Pedology Offprint requests to: J. J. Germida     

Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant

Two plant-growth-promoting bacteria, Azospirillum brasilense Cd and Pseudomonas fluorescens 313, immobilized in 1983 in two types of alginate-bead inoculant (with and without skim-milk supplement) and later dried and stored at ambient temperature for 14 years, were recovered in 1996. The population in each type of bead had decreased, yet significant numbers survived (10⁵–10⁶ cfu/g beads). Population numbers depended on the bead type and the three independent bacterial counting methods: the conventional plate-count method, indirect enzyme-linked immunosorbent assay and the limited-enrichment technique. Both bacterial species retained several of their original physiological features. When inoculated onto wheat plants, both species colonized and produced plant-growth effects equal to those of the contemporary strain from a culture collection or to their own 1983 records. This study showed that bacteria can survive in alginate inoculant over long periods. Received: 1 May 1998 / Received revision: 24 August 1998 / Accepted: 3 September 1998     

Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years <Emphasis Type="Italic">in vitro</Emphasis>

Summary Micropropagated grape (Vitis vinifera L.) cv. Arka Neelamani cultures showed a decline in root and shoot growth performance after 6–7 yr of continuous in vitro culture. Indexing the culture medium using nutrient agar or 523 bacteriological medium (Viss et al., 1991) revealed covert bacteria in 75–100% cultures. Testing the tissue from different parts of in vitro plantlets on nutrient agar showed bacteria comprising of six or more morphotypes in 100% of root and collar tissue samples but less frequently in stem segments. The shoot tips had the lowest incidence of bacterial association. The whole shoots treated with NaOCl (4% chlorine) or HgCl₂ (0.1%) showed endophytic bacterial survival. Culturing the HgCl₂-treated (5 min) shoot tips on antibiotic overlaid medium (1 ml of 50 mg l⁻¹ gentamycin and/or cefazolin) in culture tubes (150×25 mm) for 1 mo. facilitated the cleansing of cultures with 75% recovery of contaminant-free shoots as monitored through indexing for the next 2 yr. Repeated indexing of medium and tissue from various plant parts during the first two to four subculture cycles following antibiotic treatment was instrumental in reliably identifying clean cultures and preventing bacterial re-emergence. Antibiotic incorporation in the medium was detrimental to grape microcuttings. Bacteria-freed cultures showed 80–100% rooting and a high number of plantlets that could be acclimatized. The plants put in the field after 8 yr of active micropropagation showed some juvenile characteristics initially, which disappeared in 6–8 mo., and the pruned shoots showed flowering and bunch development within 1 yr of field planting. This indicated the feasibility of keeping grape plants in vitro for long periods if covert microbes were eliminated.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles. Part II. Cleavage and Uptake of Carbohydrates

Abstract The bacterial colonization and development of the ectoenzymatic glucosidase activity and glucose uptake were followed together with bacterial growth (measured as thymidine incorporation) in laboratory experiments, using phytoplankton-derived particles incubated in rolling tanks. Bacterial colonization of the particles was rapid. In the particles, bacterial turnover rates (production/biomass) were low (0.02 to 0.14 d⁻¹). In the ambient water, turnover rates increased from 0.1 d⁻¹ to 23.3 d⁻¹, until the end of the experiment. In the control, lacking any particles, turnover of bacteria ranged from 0.3 to 7.6 d⁻¹. Similarly, glucose uptake rates, per bacterium, were 1 to 2 orders of magnitude lower for particle-attached bacteria than for their free-living counterparts. Generally, K_m values for glucosidase activity declined, over the incubation period, in particles and free-living bacteria until 168 h, and slightly increased, thereafter, to values of approximately 0.1 μM. Particle-attached bacteria exhibited significantly lower uptake rates of both thymidine and glucose, per bacterium, throughout the incubation. The per-cell ectoenzymatic activity was similar in particle-associated and free-living bacteria during the initial phase of the experiment, but was significantly higher after ≈200 h. Dissolved total (TCHO), as well as monomeric carbohydrates (MCHO), declined continuously in both particles and ambient water; they remained constant in the control; TCHO comprised about 50% of the dissolved organic carbon (DOC) in the particles. In ambient water TCHO contribution to DOC varied, with only one exception, between 25 and 45%; and in the control, between 20 and 50%. The shift detectable in the relation between ectoenzymatic activity and uptake of glucose between free-living and attached bacteria over the incubation period may reflect changes in the physiological status of the bacteria. Received: 3 February 1997; Accepted: 6 November 1997     

Antiangiogenic, Antimicrobial, and Cytotoxic Potential of Sponge-Associated Bacteria

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 μg ml⁻¹ and 100% activity at 10 and 20 μg ml⁻¹ concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the α subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.     

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes

Summary The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2–3 mm³) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700 U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1×10⁴ cells/cm². These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Enhancing pozzolana colonization by As(III)-oxidizing bacteria for bioremediation purposes

The colonization of pozzolana by an As(III)-oxidizing bacterial consortium was monitored from the first hours of bacterial adhesion to 6 weeks of development under fed-batch conditions, using adapted ultrasonic dislodging and crystal-violet staining procedures to determine the biofilm adhering to the complex surfaces. The effect of temperature, arsenic concentration, and presence or absence of yeast extract (YE) on the amount of biofilm biomass and on the As(III)-oxidation were assessed to test the biofilm’s resilience and optimize the colonization. Fed-batch cultures allow twice as much pozzolana colonization as that obtained under batch conditions. In addition, As(III) oxidation and the quantities of biomass under fed-batch culture conditions were the same at 14°C and 25°C. Whereas YE improves (+150%) bacterial adhesion during the first 2 h, its impact in the longer term appears to be less significant—biofilm formation in presence of YE after 5 weeks was no greater than biofilm formation in the absence of YE. Finally, YE involves a drastic (−70%) decrease of As(III) oxidation. Preliminary tests for drinking-water bioremediation revealed the ability of Chéni Arsenic Oxidizing 1 biofilms to remain and retain As(III) oxidation activity at low As(III) concentrations (50 μg l⁻¹).     

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10⁻¹¹ to 10⁻¹² m s⁻¹) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20–30 ka BP), a disturbed interfacial zone (20–30 ka BP), and a Cretaceous clay aquitard (71–72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and thenarH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (¹⁴C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.     

Metabolic changes associated with biofilm formation in an undisturbed Mediterranean stream

Respiratory activity (ETS), ectoenzymatic activity (-glucosidase and -xylosidase) and photosynthetic ¹⁴C-bicarbonate incorporation in the biofilm were measured in a shaded stream during a colonization sequence (43 days) on artificial substrates (unglazed clay tiles) and compared with older (aged) tiles. In the first five days bacterial densities and ectoenzyme activities showed a sharp increase. After two weeks, algal density, chlorophyll and productivity increased moderately. Chlorophyll did not reach similar values to those of the aged biofilms until the last day of colonization. Photosynthetic activity seemed to be relevant to the heterotrophs metabolism during substrate colonization, as could be deduced from the significant correlation between -glucosidase and ¹⁴C-bicarbonate incorporation, algal cell densities and chlorophyll. Respiratory activity (ETS) decreased in the older biofilms, accordingly to their higher algal and bacterial density. Younger biofilms (up to 8 days old) showed higher ETS activity per cell, indicating a fast response of microorganisms to substrate availability.     

Nano silver: a novel nanomaterial for removal of bacterial contaminants in valerian (Valeriana officinalis L.) tissue culture

Bacterial contamination is a serious problem in plant tissue culture procedures. An experiment was conducted to evaluate the potential of nano silver (NS) to remove bacterial contaminants of valerian nodal explants. This experiment was conducted as a completely randomized design in a factorial arrangement with four replications and each replicate with ten explants. Treatments involved NS at two stages (before and after surface sterilization along with control) with three rates (25, 50 and 100 mg l⁻¹) at three times of soaking (30, 60 and 180 min). Explants were cultured on MS medium supplemented with 5 mg l⁻¹ Kin and 0.1 mg l⁻¹ NAA. Results showed that using 100 mg l⁻¹ of NS solution after surface sterilization resulted in the highest percentage (89%) of disinfected explants. Nano silver solution did not affect the characters measured. On the basis of the data obtained in this experiment, it was concluded that NS had a good potential for removing of the bacterial contaminants in plant tissue culture procedures. As this is the first report on application of NS in in vitro culture techniques, further investigations on other plant species are needed to clarify the effectiveness of NS for the removal of bacterial contaminants in tissue culture of other crops.     

Extracellular deoxyribonuclease production by a thermophile, Streptomyces thermonitrificans

A thermophilic bacterial strain, Streptomyces thermonitrificans, produced high levels of extracellular deoxyribonuclease (DNase) when grown on NBG medium (containing 1% peptone, 0.3% beef extract, 1% glucose and 0.5% NaCl). Maximum DNase activity (140 U ml⁻¹) was obtained, in 24 h, when the culture was grown on modified NBG medium (containing 1.3% beef extract, 1% glucose, 0.5% NaCl and 50 μM Mn²⁺ at 45°C. The crude enzyme showed higher activity on native DNA than on sonicated and heat denatured DNA. Moreover, addition of Mn²⁺ in the assay mixture resulted in a significant stimulation (10–15 fold) of the enzyme activity. Received 24 November 1998/ Accepted in revised form 25 April 1999     

Inactivation of the Antibacterial and Cytotoxic Properties of Silver Ions by Biologically Relevant Compounds

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag⁺ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag⁺ and thiols are added in a 1∶1 ratio because the reaction of Ag⁺ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings.     

Quantification and Localization of Bacteria in Plant Tissues Using Quantitative Real-Time PCR and Online Emission Fingerprinting

In order to quantify and localize specific bacterial target genes in plant tissue, this project has generated relevant new insights in the combined application of quantitative real-time PCR in parallel with the in situ PCR + probe-hybridization and online emission fingerprinting using LSM 510 META. After designing an Enterobacter radicincitans species-specific probe, introduced bacterial cells were monitored in growing plant parts and their colonization behaviour was examined in relation to the native bacterial community. For this purpose, the plant growth-promoting rhizobacterial (PGPR) strain Enterobacter radicincitans was applied to Brassica oleracea plants in increasing inoculum concentrations 10⁷, 10⁸ and 10⁹ cells per plant. Inoculation of 10⁹ E. radicincitans cells per plant to Brassica oleracea leaves and roots resulted in significant increases of root, leaf and tuber growth. Total bacterial cell numbers were estimated using quantitative real-time PCR to be between 10⁷ and 10⁹ cells g⁻¹ fresh leaf weight and about 10⁸ cells g⁻¹ fresh root weight of Brassica oleracea plants. Using quantitative real-time PCR, a significant colonization of Brassica oleracea leaves and roots with E. radicincitans cells was measured. Roots were colonized with a density of 10⁷ cells g⁻¹ fresh root weight up to at least 14 days after inoculation. That is equivalent to a proportion of E. radicincitans 16S rDNA-gene copy numbers compared to the total bacterial communities of about 10–16%. Online emission fingerprinting established that the introduced bacteria proliferated on and inside the root and that they colonized the intercellular spaces of the root cortex layer. Hence, E. radicincitans was able to successfully compete with the native bacterial population.