Characterization of bacteria contaminating tissue cultures of apple rootstock 'YP'

Meristem-tip cultures of apple rootstock 'YP' were started at different times of the year over a period of 2 years and the contamination of the cultures was monitored during five subcultures. Bacterial contaminants were isolated to pure cultures, identified by the API test system and appropriate additional tests, and the sensitivity of the most common isolates to different antibiotics was determined. Of the 216 strains isolated from the initiation cultures, 78% were pseudomonads, coryneforms or enterobacteria. Only three bacterial contaminants were found at the multiplication stage. A greater part of the contaminants were likely to originate from the stock plant. Rifampicin (at 50–200 mg 1^-1) and cefotaxime (at 250–1500 mg 1^-1) were found to be bactericidal against many isolates, but differences between species and strains were found.     

Environmental microbial contamination in a stem cell bank

AIM: The aim of this study was to evaluate the main environmental microbial contaminants of the clean rooms in our stem cell bank. METHODS AND RESULTS: We have measured the microbial air contamination by both passive and active air sampling and the microbial monitoring of surfaces by means of Rodac plates. The environmental monitoring tests were carried out in accordance with the guidelines of European Pharmacopeia and US Pharmacopeia. The micro-organisms were identified by means of an automated system (VITEK 2). During the monitoring, the clean rooms are continually under good manufacturing practices specifications. The most frequent contaminants were Gram-positive cocci. CONCLUSIONS: The main contaminants in our stem cell bank were coagulase-negative staphylococci and other opportunistic human pathogens. In order to assure the levels of potential contamination in both embryonic and adult stem cell lines, a continuous sampling of air particles and testing for viable microbiological contamination is necessary. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first evaluation of the environmental contaminants in stem cell banks and can serve as initial evaluation for these establishments. The introduction of environmental monitoring programmes in the processing of stem cell lines could diminish the risk of contamination in stem cell cultures.     

Direct isolation of mycoplasmas and acholeplasmas from sera and kidneys of calves.

Kidneys and sera of calves from various farms and primary kidney tissue cultures were tested for mycoplasma and acholeplasma contamination. Altogether 24 strains belonging to Mycoplasma arginini and Acholeplasma axanthum were isolated from tissue cultures, kidneys and sera. The same species were detected from lungs and peribronchial lymph nodes of calves, together with A. laidlawii, A. modicum and M. bovirhinis species. There was a close correlation between the mycoplasma content of tissue cultures, sera and kidneys and the clinical picture observed in the farm, as well as between the quality of tissue cultures and the mycoplasma content of organs, sera and tissue cultures.     

Biodiversity of cultivable psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea, Antarctica)

A set of 146 Antarctic marine isolates from the Ross Sea was characterized by a combination of molecular techniques in order to determine the degree of inter- and intraspecific variability. Isolates were analyzed by amplified rDNA restriction analysis (ARDRA) using the tetrameric enzyme AluI, resulting in 52 different groups, corresponding to at least 52 different bacterial species, indicating a high degree of interspecific variability. The phylogenetic position of bacteria belonging to some ARDRA groups was obtained by sequencing of 16S rDNA. Random amplified polymorphic DNA (RAPD) analysis, carried out on the largest ARDRA groups, revealed a high intraspecific genetic variability, too. The analysis of plasmid content revealed the existence of horizontal gene transfer between strains belonging to the same and to different species. A comparison of the whole body of morphological, physiological and biochemical data was finally carried out.     

Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes

Fourier transform infrared (FT-IR) spectroscopy is a convenient physico-chemical technique to investigate various cell materials. Bacteria of class Mollicutes, identified by conventional methods, as Mycoplasma, Acholeplasma and Ureaplasma genera were characterized using this method. A data set of 74 independent experiments corresponding to fourteen reference strains of Mollicutes was examined by FT-IR spectroscopy to attempt a spectral characterization based on the biomolecular structures. In addition to the separation of Mollicutes within the lipidic region into five main clusters corresponding to the three phylogenetic groups tested, FT-IR spectroscopy allowed a fine discrimination between strains belonging to the same species by using selective spectral windows, particularly in the 1200-900 cm(-1) saccharide range. The results obtained by FT-IR were in good agreement with both taxonomic and phylogenetic classifications of tested strains. Thus, this technique appears to be a useful tool and an accurate mean for a rapid characterization of Mollicutes observed in humans.     

Intraspecific Differentiation of Vibrio vulnificus Biotypes by Amplified Fragment Length Polymorphism and Ribotyping

The intraspecific genomic relatedness of 80 Vibrio vulnificus isolates, 44 of biotype 1 and 36 of biotype 2, from different geographic origins and sources was evaluated by ribotyping and AFLP (amplified fragment length polymorphism) fingerprinting. Ribopatterns of DNAs digested with KpnI and hybridized with an oligonucleotide complementary to a highly conserved sequence in the 23S rRNA gene revealed up to 19 ribotypes in the species, which were different for the two biotypes. Sixteen different ribotypes were found within biotype 1 strains from clinical and environmental sources, and only three, recovered mainly from diseased eels, were found within biotype 2. Within this biotype, 96% of the strains showed the same ribopattern. The closest similarity was shown by the strains coming from the same eel farm, irrespectively of biotype. AFLP fingerprints obtained by selective PCR amplification of HindIII-TaqI double-restricted DNA fragments exhibited a strain-specific pattern which allowed the finest differentiation of subgroups within the eel-pathogenic isolates sharing the same ribopattern. Both techniques revealed good genetic markers for intraspecific differentiation of V. vulnificus. Ribotyping clearly separated the eel-pathogenic strains from the clinical and environmental isolates, whereas AFLP enabled the monitoring of individual strains and therefore constitutes one of the most discriminative tools for epidemiological and ecological studies.     

Comparative study of biological hydrogen production by pure strains and consortia of facultative and strict anaerobic bacteria

In this paper, a simple and rapid method was developed in order to assess in comparative tests the production of binary biogas mixtures containing CO₂ and another gaseous compound such as hydrogen or methane. This method was validated and experimented for the characterisation of the biochemical hydrogen potential of different pure strains and mixed cultures of hydrogen-producing bacteria (HPB) growing on glucose.The experimental results compared the hydrogen production yield of 19 different pure strains and sludges: facultative and strict anaerobic HPB strains along with anaerobic digester sludges thermally pre-treated or not. Significant yields variations were recorded even between different strains of the same species by i.e. about 20% for three Clostridium butyricum strains. The pure Clostridium butyricum and pasteurianum strains achieved the highest yields i.e. up to 1.36 mol H₂/mol glucose compared to the yields achieved by the sludges and the tested Escherichia and Citrobacter strains.     

Bacterial contaminants of micropropagated plant cultures

Bacterial contaminants of micropropagated plant cultures were isolated and characterized with standard bacteriological tests and appropriate API strips. Results obtained were analysed by the API identification software. Of 198 bacterial strains isolated from nine plant species, 90% were identified as Bacillus, Enterobacter, Micrococcus, Staphylococcus, Pseudomonas or Lactobacillus species. Possible sources of contamination are discussed.     

Uracil phosphoribosyl transferase activity of mycoplasma and infected cell cultures

Summary Human H. Ep-2 and mouse 3T6 cells infected byMycoplasma hyorhinis showed an increase in [³H]uracil uptake and a more than 20-fold increase in the activity of uracil phosphoribosyltransferase (UraPRT). Uninfected cell cultures gave background levels of this enzyme activity. A survey of 16 strains of mycoplasma showed 13 to possess UraPRT activity. Rabbit kidney cells (RK13) were infected with eight different strains of four mycoplasma species known to be common cell culture contaminants. Seven of the eight cell cultures showed elevated UraPRT activities four days after infection. This enzyme activity may be of value in monitoring cell cultures for mycoplasma and aid in classification. This work was supported by Contract NO1-CP-53530 with the National Cancer Institute, National Institutes of Health, and Contract FDA 74-41 of the Food and Drug Administration, Bethesda, Maryland 20014.     

Effect of non-target cells on the sensitivity of the PCR for Escherichia coli O157 : H7

The fatty acid composition from mycelia of Streptomyces hygroscopicus strains was studied. A significant proportion of C_{18 : 2} was found in cultures. High levels of C_{16 : 0}, iso-C_{16 : 0} and C_{18 : 1} were also detected in all S. hygroscopicus strains. The different representatives of S. hygroscopicus had almost the same proportion of unsaturated fatty acids. Certain shifts in the amount of iso, anteiso and straight-chain fatty acids in some cultures were revealed. This might be explained by the adaptation capability of strains belonging to one species to form a variety of available fatty acids determined by particular cell membrane composition favouring certain antibiotic biosynthesis.     

Risk factors and transmission routes of the causative agents of campylobacteriosis in commercial poultry plants]

The study, carried out in two regions of the USSR and aimed at estimation of the contamination of products supplied by industrial poultry complexes (IPC), revealed that the contamination of these products was closely related to the Campylobacter contamination of the personnel of IPC. The causes of high Campylobacter contamination of the products of IPC at all technological stages of their production are described. The species, serovars and biovars of Campylobacter strains isolated from different sources were determined, which made it possible to carry out the specific and intraspecific differentiation of these strains.     

Characterization and control of endophytic bacterial contaminants in in vitro cultures of Piper spp., Taxus baccata subsp. wallichiana, and Withania somnifera

Bacterial contamination is a serious problem that causes severe loss of in vitro grown cultures of a number of plants. This problem becomes even more acute if the bacterial contamination is of endophytic origin. In such cases, identification and characterization of the contaminants is essential for achieving specific control of the contaminants through selective use of antibiotic agents, especially if the routinely used contamination control methods practiced elsewhere in tissue culture studies are ineffective. Such is the case with the bacterial contamination observed in the present study. The five endophytic bacteria associated with Piper nigrum and Piper colubrinum, four endophytic bacteria associated with Taxus baccata subsp. wallichiana, two endophytic bacteria associated with Withania somnifera, and two bacteria common to all these plant species were isolated and characterized based on morphological and biochemical tests. Their taxonomic positions based on similarity indices were determined. A control strategy against these bacteria has been developed based on bacteriostatic or bactericidal actions of 12 antibiotics at three different concentrations by solid and liquid antibiogramme assays.     

Correlating species and spectral diversities using hyperspectral remote sensing in early‐successional fields

Advances in remote sensing technology can help estimate biodiversity at large spatial extents. To assess whether we could use hyperspectral visible near‐infrared (VNIR) spectra to estimate species diversity, we examined the correlations between species diversity and spectral diversity in early‐successional abandoned agricultural fields in the Ridge and Valley ecoregion of north‐central Virginia at the Blandy Experimental Farm. We established plant community plots and collected vegetation surveys and ground‐level hyperspectral data from 350 to 1,025 nm wavelengths. We related spectral diversity (standard deviations across spectra) with species diversity (Shannon–Weiner index) and evaluated whether these correlations differed among spectral regions throughout the visible and near‐infrared wavelength regions, and across different spectral transformation techniques. We found positive correlations in the visible regions using band depth data, positive correlations in the near‐infrared region using first derivatives of spectra, and weak to no correlations in the red‐edge region using either of the two spectral transformation techniques. To investigate the role of pigment variability in these correlations, we estimated chlorophyll, carotenoid, and anthocyanin concentrations of five dominant species in the plots using spectral vegetation indices. Although interspecific variability in pigment levels exceeded intraspecific variability, chlorophyll was more varied within species than carotenoids and anthocyanins, contributing to the lack of correlation between species diversity and spectral diversity in the red‐edge region. Interspecific differences in pigment levels, however, made it possible to differentiate these species remotely, contributing to the species‐spectral diversity correlations. VNIR spectra can be used to estimate species diversity, but the relationships depend on the spectral region examined and the spectral transformation technique used.     

A risk assessment study of plant genetic transformation usingAgrobacterium and implications for analysis of transgenic plants

Agrobacterium transformation systems forBrassica, Solanum andRubus, using carbenicillin, cefotaxime and ticaracillin respectively to eliminate contamination, were examined for the presence of residualAgrobacterium. The results indicated that none of the antibiotics in question, succeeded in eliminatingAgrobacterium and the contamination levels increased in explants from 12 to 16 weeks to such an extent thatSolanum cultures senesced and died. This may be due to the fact that four times the Minimum bactericidal concentration values (concentration to be used for elimination of contaminants in culture), for the three antibiotics, were higher than the concentrations employed in the culture medium. Contamination in shoot material decreased over 16 to 24 weeks possibly due to bacteriostatis and the use only of the apical node for further culture. The presence of the binary vector was also noted under non-selective conditions, even up to 6 months after transformation, where approx. 50% of contaminated material still harboured bacterial cells with the binary vector at levels of approx. 10₇ Colony forming units per gram.     

Discrimination of enterobacterial repetitive intergenic consensus PCR types of Campylobacter coli and Campylobacter jejuni by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm(-1) (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.     

Utilisation of differential killer toxin sensitivity patterns for fingerprinting and clustering yeast strains belonging to different genera

The differential killer sensitivity of 103 yeast cultures belonging to 12 species (genera Debaryomyces, Kluyveromyces, Saccharomyces, and Zygosaccharomyces), all previously taxonomically certified by nDNA-nDNA reassociation, against a given panel of 39 killer yeasts was used as a fingerprinting tool. All strains, with the only exception of eight cultures belonging to the species Zygosaccharomyces bailii, were characterised by a specific, individual sensitivity pattern (killer formula). Cluster analysis of binary sequences based on killer sensitivity of strains belonging to different genera is presented and discussed.     

Comparative evaluation of different preservation methods for cyanobacterial strains

Maintaining pure cultures using preservation methods is of high importance for biotechnological purposes. However, preservation does not necessarily guarantee the genetic stability of these cultures. Therefore, preservation methods are currently needed to assure viability as well as genetic, physiological, and morphological integrity across storage periods. In this study, preservation of five isolates from the microalgae and cyanobacteria collection of the Plant Biology Department, Federal University of Viçosa, Minas Gerais, Brazil was investigated via monthly analyses of cell viability, biomass recovery, and contaminant concentrations over a period of 120 days. Lyophilization was adequate for both heterocystous cyanobacteria and other strains that were able to differentiate hormogones or to synthesize thick layers of exopolysaccharides. Lyophilization was also able to maintain cultures with low levels of contaminants. Dimethyl sulfoxide was relatively efficient, though some of the strains were susceptible to its cytotoxic effects. Our results demonstrated that cryopreservation with glycerol was the most efficient method. The ability to routinely preserve cyanobacterial strains reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. The results obtained in this study are therefore discussed in the context of the efficiency of the methods and the current need to develop suitable methods for maintenance of cyanobacterial collections.     

Intra- and interspecific variation of cuticular hydrocarbon composition in two Ectatomma species (Hymenoptera: Formicidae) based on Fourier transform infrared photoacoustic spectroscopy

We have been able to discriminate different castes and sexes of ants in the same colony by measuring cuticular hydrocarbon levels with Fourier transform infrared photoacoustic spectroscopy, compared by canonical discriminant function analysis. We have now applied this methodology to various colonies of two species of ants of the genus Ectatomma in the Brazilian Cerrado. There were clear interspecific differences in cuticular hydrocarbons of these ants, with a small intraspecific variation. The differences between colonies were greater in E. brunneum than in E. vizottoi. Genetic differences among the colonies and species were well estimated by Fourier transform infrared photoacoustic spectroscopy and statistical analyses.     

Terrestrial and aquatic bryophytes as monitors of environmental contaminants in urban and industrial habitats

The widespread distribution of bryophytes and the tolerance of many species to certain contaminants has led to their use for monitoring purposes. Early this century, changes in the distribution of mosses in urban habitats indicated deteriorating air quality; alterations in species composition in rivers has reflected changing water quality. The main focus in the past 10 to 20 years has been on measuring levels of contaminants in both terrestrial and aquatic bryophytes. Concentrations of metals, organic chemicals, radionuclides and derivatives of acidic gases have been widely reported in species from contaminated and background areas. More rarely, physiological and biochemical parameters are monitored. This paper describes the approaches which have been used and results obtained from monitoring bryophytes in urban and industrial habitats. Data are reviewed from a range of countries illustrating the increasing interest in low cost methods for monitoring contamination.     

Rapid strain classification and taxa delimitation within the edible mushroom genus Pleurotus through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy

Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the identification of bacteria and yeasts, but only to a limited extent for discriminating specific groups of filamentous fungi. In the frame of this study, 73 strains - from different associated hosts/substrates and geographic regions - representing 16 taxa of the edible mushroom genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated on the basis of presence/absence of specific absorbance peaks combined with cluster analysis, demonstrated that the spectral region 1800-600 cm(-1) permitted clear delimitation of individual strains into Pleurotus species. In addition, closely related species (e.g., Pleurotus ostreatus and Pleurotus pulmonarius) or taxa of the subgenus Coremiopleurotus demonstrated high similarity in their absorbance patterns, whereas genetically distinct entities such as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy differences. When specific regions (1800-1700, 1360-1285, 1125-1068, and 950-650 cm(-1)) were evaluated in respect to the absorbance values demonstrated by individual strains, it was evidenced that this methodology could be eventually exploited for the identification of unknown Pleurotus specimens with a stepwise process and with the aid of a dichotomous key developed for this purpose. Moreover, it was shown that the nature of original fungal material examined (mycelium, basidiomata, and basidiospores) had an effect on the outcome of such analyses, and so did the use of different mycelium growth substrates. In conclusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efficient solution for the classification of pure cultures from closely related mushroom species.