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PubMed2005

The prokaryotic diversity of biological soil crusts in the Sonoran Desert (Organ Pipe Cactus National Monument, AZ).

We studied prokaryotic community structure and composition in biological soil crusts (BSCs) from the Sonoran Desert, and their variability over space and time, using statistically analyzed, PCR-based molecular surveys of environmental 16S rRNA genes. Four sites, tens of km apart, were sampled, 3 times over a 1 year period, collecting 10 duplicate samples every 50 m in each site. Denaturing gradient gel electrophoresis (DGGE) revealed communities much less diverse than those of typical soil assemblages, displaying dominance of some bacterial types. No differences in crust microbial diversity or composition were detected between crusts under plant canopies and those in plant interspaces, indicating a likely crust independence from higher plant resources. However, statistically significant variability with space and time could be detected, and samples within a site were more similar than samples between sites. Both temporal and spatial variability in community composition involved non-dominant members of the community. Extensive sequencing and phylogenetic analysis revealed a large array of bacterial types, many novel. The most common included members of Cyanobacteria, Proteobacteria, Actinobacteria and Acidobacteria. Bacteriodetes, Chloroflexi and Gemmatimonadetes were not seen in high numbers, but were present in all sites, and Deinococci were also detected. Archaea were present, but as minor components. Sonoran BSC communities were distinct in rough compositional character from those in bulk arid soils or agricultural soils, and contained reoccurring, uncultured microbes.

Archaea↗

PubMed2004

Photosynthesis in sediments determined at high spatial resolution by the use of microelectrodes.

The present study investigated photosynthetic rates and their regulation by light within the upper 5mm of sediment in a tidal area of Niida River in Hachinohe, Japan. Steady-state concentration profiles of O(2), NH(4)(+), NO(2)(-), H(2)S, and pH in the sediment were measured with microelectrodes. Microzonation of O(2) respiration, denitrification and SO(4)(2-) reduction was found in the sediment. When light intensities exceeded 1050 micromol photons/m(2)/s, net photosynthetic activity was detected in the upper 0.5mm of the microbial mat colonizing on the sediment surface in the tidal area. In contrast, gross photosynthetic activity was detected in the upper 1.0mm of the microbial mat at 1900 micromol photons/m(2)/s. As light intensity increased, the net photosynthetic rate and O(2) penetration depth increased. The maximal net photosynthetic rate and O(2) penetration depth were 6.1 micromol O(2)/cm(3)/h and 2.2mm, respectively, at 1900 micromol photons/m(2)/s. Net photosynthetic rates in the microbial mat in the tidal area were lower than in the upstream sediment. The analysis of continuous O(2) concentration measurements in different layers of the microbial mat during artificial light-dark cycles demonstrated that the photosynthetic activity response to changes in light intensity was extremely fast (a few seconds) and the O(2) concentration in the microbial mat became stable within 200s. The measurement of physical and chemical parameters in river water revealed that the study site was relatively polluted and sunlight intensity significantly fluctuated temporally. These results suggested that the in situ microbial processes occurring in the sediment fluctuated in accordance with periodic fluctuations in sunlight intensity.

Bacteria↗

PubMed1988

Rapid quantification of bacterial and fungal growth in burn wounds: biopsy homogenate Gram stain versus microbial culture results.

A prospective analysis of 370 burn wound biopsies was done to correlate Gram-stain results from biopsy homogenates with quantitative culture results. The number of bacteria seen in a total of 10 oil immersion microscope fields of Gram-stained homogenates was correlated with significant microbial growth (1 x 10(5) organisms/gram of tissue) of the same biopsy homogenate plated on trypticase soy agar. Of the biopsies examined, Gram-negative rods were present in 36.8 per cent, Gram-positive cocci in 49.7 per cent and yeast in 15.9 per cent. Mixtures of organisms were present in 24.3 per cent. When Gram stains showed one or more organisms per oil immersion microscope field, the correlation with significant microbial growth was 94.5 per cent or more. When five or more organisms were seen per field, the correlation with significant growth became 97 per cent or greater. When no organism was seen on Gram stain, the cultures grew significant numbers of organisms 19.1 per cent of the time or less. This false-negative rate was considered to be high. It is believed, however, that this method of early detection of significant burn wound microbial growth may prove to be valuable in the management of severely burned patients.

Agar↗

PubMed1999

Bacterial function and community structure in reactors treating biopolymers and surfactants at mesophilic and thermophilic temperatures.

Microbial communities capable of degrading biopolymers and surfactants typically found in graywater were selected in continuous-flow bioreactors operated at 30, 44, 53, or 62 degrees C. The effect of temperature upon microbial activity and community composition was determined. Microbial respiration of the organic components of the medium (including linear alkylbenzene sulfonate) was detected in samples from each reactor. The microbial community in each reactor was adapted to the operating temperature. Nucleic acid-based analyses of community composition showed that distinct consortia were present at each temperature. Community complexity was inversely related to temperature. The specific maintenance rate was twofold higher at 62 degrees C than at the lower temperatures. Under starvation conditions, microbes in the 62 degrees C system lost membrane integrity 30- to 100-fold faster than microbes at lower temperatures.

Journal Article↗

PubMed1987

[Detection of the antigens of the causative agent of brucellosis by an immunoenzyme method].

The conditions of the enzyme immunoassay for the detection of Brucella antigens have been selected, making it possible to detect these antigens both in solutions and in biological material within 3-4 hours. In guinea pigs infected with B. abortus 99 in a dose of 1,000 microbial cells, brucellar antigen has been detectable in the organs and blood serum of the animals as early as 24 hours after infection. This assay, if carried out under the optimal conditions, detects soluble brucellar polysaccharide antigen at a concentration of 1 ng/ml and Brucellae at a concentration of 5 X 10(4) microbial cells/ml in the presence of 200-fold surplus of other bacterial cells.

Animals↗

PubMed1986

Clinical comparison of a new automated infrared blood culture system with the BACTEC 460 system.

A new blood culture instrument, the BACTEC (Johnston Laboratories, Inc., Towson, Md.) NR-660, which utilizes infrared detection of carbon dioxide from microbial metabolism, was compared with the radiometric BACTEC 460 system. There were 1,554 isolates from 18,785 paired aerobic blood cultures. Of these isolates, 1,303 were isolated from the radiometric 6B medium, and 1,259 were isolated from the NR6A medium (P = 0.06). Analysis of the data indicated no significant differences in recovery when any individual species was considered. When organisms were considered as groups, there were no significant detection differences for gram-negative bacilli, yeasts, or anaerobes. For gram-positive cocci in aerobic medium, the BACTEC 460 detected 84.3% of the total isolates, and the BACTEC NR-660 detected 79.7% (P = 0.04). There were 891 isolates from 13,983 paired anaerobic blood cultures. Of these isolates, 725 were recovered from the radiometric 7D BACTEC medium, and 723 were recovered from the NR7A BACTEC medium (P greater than 0.9). In the anaerobic media there was no significant difference in detection of any organism group, including the gram-positive cocci. When the results of the aerobic and anaerobic media were combined, there was equivalence between the two systems for the detection of gram-positive cocci (P greater than 0.2) and other organism groups. When the ability to detect septic episodes was compared, there was no significant difference for any organism group (P = 0.12). For aerobic media, the mean times for detection were 30.5 and 29.5 h for the BACTEC 460 and NR-660, respectively. For anaerobic media, the mean times for detection were 39.8 and 41.6 h for the BACTEC 460 NR-660, respectively. Compared with the BACTEC 460, the BACTEC NR-660 system had a greater ease of operation, faster test cycle, computerized data base, and equally rapid detection of positive cultures.

Bacteria↗

PubMed2000

Comparison of colony lift with direct spotting methods of blot preparation on the effect of colony hybridization in the detection of environmental organisms.

Nucleic acid probes are used on site to detect or to identify individual microbial cells without cultivation. This molecular technique can avoid some limitations of traditional identification methods including time consuming and imprecise. This study examined the factors affecting colony hybridization and compared the effectiveness of membrane prepared by colony lifting with direct spotting procedures using the universal probe Eub 338. The results of hybridization varied depending on the type of colony morphology. For dry and rough colonies, colony hybridization was not suitable for detecting Acinetobactersp. (CK2A, CK2B), Alcaligenes sp. (TH11 B), Xanthomonas sp. (TH7B), Arthrobacter globiformis (CCRC 10598) and Microbacterium sp. (CCRC 11036). Colonies of Acinetobacter sp. (CCRC 15425) and Alcaligenes spp. (CCRC 10828, H) on agar and membrane were thick and raised, and their detection signals of hybridization were diffused or blank. Colonies of Alcaligenes sp. (CM7A, ANV2) and Acinetobacter sp. (ANV8) isolated from the sludge of biological processes treating ABS wastewater were flat and smooth, and their hybridization signals were clear. For those strains suitable for colony hybridization, the colony blots prepared by colony lift and direct spotting procedures gave the same sensitivity for colony hybridization.

Bacteria↗

PubMed2001

Confocal laser scanning microscopy as a tool to determine cyanobacteria biomass in microbial mats.

We have developed a method based on confocal laser scanning microscopy for detection and quantification of cyanobacteria from the Ebro Delta microbial mats. Cyanobacteria play a major role as primary producers in microbial mats; it is difficult, however, to apply classical methods to estimate their biomass because they establish strong interactions with detritic particles. The protocol described here allows the localization of individual cells or filaments with micrometre precision without the need to either manipulate or stain the samples. This method is suitable for studying biomass 'in situ' in microbial mats.

Biomass↗

PubMed2006

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms.

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.

Colorimetry↗

PubMed1995

Microbiological assessment of an improved stannous fluoride dentifrice.

Recently, a new stabilized stannous fluoride (SnF2) dentifrice (SSF) has been developed. The aim of the present work was to examine the antimicrobial activity of SnF2, and to assess the long-term microbial safety of this dentifrice in a series of in vitro and clinical evaluations. Results of in vitro time-kill experiments with representative oral bacteria demonstrated that SnF2 exerts broad antimicrobial activity against both Gram-positives and Gram-negatives and, in particular, has potent activity against Streptococcus mutans. Sixty-eight subjects participated in a nine-day plaque regrowth clinical study to assess the short-term antiplaque effect of SSF. The results revealed no significant differences from the negative control, suggesting that SnF2 does not detectably or directly alter plaque microbial viability or composition. Separately, evaluation of microbial safety in a subgroup of 120 subjects participating in a six-month clinical efficacy and safety trial found no significant ecological shifts between SSF and the negative (NaF dentifrice) control among 11 supragingival microbial populations examined. The potential for development of bacterial resistance to SnF2 was assessed under both in vitro and clinical conditions. In a rigorous assessment of the ability of bacterial populations to develop either phenotypic or genotypic resistance to SnF2, representative bacteria were exposed to continuous sub-lethal concentrations of SnF2 in a laboratory chemostat for at least 9 days. Results of time-kill experiments on exposed populations revealed no significant changes in susceptibility despite exposure of over 10(12) bacteria. Based on typical spontaneous mutation rates of 10(-6) to 10(-8), these results suggested that the potential for bacteria to develop resistance to SnF2 is low. Evaluation of susceptibility to SnF2 to over 800 bacterial isolates obtained over the course of the six-month clinical trial corroborate the in vitro findings, revealing no changes in susceptibility suggestive of development of resistance to SnF2 is a microbiologically safe agent for oral use and support separate clinical observations demonstrating the safety and efficacy of this stabilized SnF2 dentifrice.

Actinomyces↗

PubMed1999

Seasonal variations in microbial populations and environmental conditions in an extreme acid mine drainage environment.

Microbial populations, their distributions, and their aquatic environments were studied over a year (1997) at an acid mine drainage (AMD) site at Iron Mountain, Calif. Populations were quantified by fluorescence in situ hybridizations with group-specific probes. Probes were used for the domains Eucarya, Bacteria, and Archaea and the two species most widely studied and implicated for their role in AMD production, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans. Results show that microbial populations, in relative proportions and absolute numbers, vary spatially and seasonally and correlate with geochemical and physical conditions (pH, temperature, conductivity, and rainfall). Bacterial populations were in the highest proportion (>95%) in January. Conversely, archaeal populations were in the highest proportion in July and September ( approximately 50%) and were virtually absent in the winter. Bacterial and archaeal populations correlated with conductivity and rainfall. High concentrations of dissolved solids, as reflected by high conductivity values (up to 125 mS/cm), occurred in the summer and correlated with high archaeal populations and proportionally lower bacterial populations. Eukaryotes were not detected in January, when total microbial cell numbers were lowest (<10(5) cells/ml), but eukaryotes increased at low-pH sites ( approximately 0.5) during the remainder of the year. This correlated with decreasing water temperatures (50 to 30 degrees C; January to November) and increasing numbers of prokaryotes (10(8) to 10(9) cells/ml). T. ferrooxidans was in highest abundance (>30%) at moderate pHs and temperatures ( approximately 2.5 and 20 degrees C) in sites that were peripheral to primary acid-generating sites and lowest (0 to 5%) at low-pH sites (pH approximately 0.5) that were in contact with the ore body. L. ferrooxidans was more widely distributed with respect to geochemical conditions (pH = 0 to 3; 20 to 50 degrees C) but was more abundant at higher temperatures and lower pHs ( approximately 40 degrees C; pH approximately 0.5) than T. ferrooxidans.

Archaea↗

PubMed2006

Cyanobacterial ecotypes in the microbial mat community of Mushroom Spring (Yellowstone National Park, Wyoming) as species-like units linking microbial community composition, structure and function.

We have investigated microbial mats of alkaline siliceous hot springs in Yellowstone National Park as natural model communities to learn how microbial populations group into species-like fundamental units. Here, we bring together empirical patterns of the distribution of molecular variation in predominant mat cyanobacterial populations, theory-based modelling of how to demarcate phylogenetic clusters that correspond to ecological species and the dynamic patterns of the physical and chemical microenvironments these populations inhabit and towards which they have evolved adaptations. We show that putative ecotypes predicted by the theory-based model correspond well with distribution patterns, suggesting populations with distinct ecologies, as expected of ecological species. Further, we show that increased molecular resolution enhances our ability to detect ecotypes in this way, though yet higher molecular resolution is probably needed to detect all ecotypes in this microbial community.

Adaptation, Biological↗

PubMed1987

[Carcinogenic and genotoxic effects of mycotoxins].

Aflatoxin B1 is the most potent hepatocarcinogen among the naturally occurring compounds. It's carcinogenic effect was confirmed by longterm-studies in several animal species. There exists detailed knowledge on the molecular mechanism of the carcinogenic effect caused by aflatoxin B1. At present there does not exist a reliable evidence that human primary hepatocellular carcinoma is caused by aflatoxins. Sterigmatocystin is a hepatocarcinogen too. The genotoxicity of sterigmatocystin likewise as of aflatoxin B1 was detected by short-term-tests with microbial systems and mammalian cells in vitro. It is disputable whether ochratoxin A, citrinin and T-2 toxin are carcinogenic or not. Diacetoxyscirpenol, zearalenone and patulin are estimated as not carcinogenic after longterm-studies with oral toxin administration. Patulin and penicillic acid are genotoxic in microbial systems respectively in mammalian cells in vitro. Luteoskyrin is carcinogenic, but there was not detected any genotoxicity.

Animals↗

PubMed1997

A retrospective analysis of microbial contaminants in outdated random-donor platelets from multiple sites.

BACKGROUND: Platelet components contaminated with bacteria are an important source of transfusion-associated bacterial sepsis. Estimates of contamination rates vary widely (0-10%) and are highly controversial. The present study, designed with stringent testing regimens, retrospectively determined the prevalence of microbial contaminants in platelets from four collection regions. STUDY DESIGN AND METHODS: During a 9-month period, outdated platelet units were assayed by spreading aliquots from the unit, and from thioglycollate broth medium inoculated with part of the unit, onto 5-percent sheep blood agar media. Cultures were examined after 72-hour incubation at 37 degrees C, and, if bacterial growth was present, the assay processes were repeated with fresh inocula. Units were considered contaminated only if repeatedly positive. RESULTS: Four (0.08%) of 4995 units sampled were contaminated, two with Corynebacterium sp. and one each with Propionibacterium acnes and Aspergillus terreus. Contaminants were present at low, subclinical levels and were detected only after amplification in thioglycollate. The contaminated units were cultured 1, 2, 3, and 7 days after expiration. CONCLUSION: Contamination rates were low and did not vary by region. The identification of A. terreus suggests the role that transfusion may play in transmitting fungal infections should be reassessed. The persistent detection of contaminated platelet units supports the need for a test to detect clinically relevant levels of microbial contaminants in blood components.

Aspergillus↗

PubMed1979

Detection of the mutagenic activity of lead chromate using a battery of microbial tests.

The potential mutagenicity of the carcinogen lead chromate was tested by the following battery of microbial tests: the Escherichia coli PolA+/PolA- survival test; the Salmonella/microsome His+ reversion assay; the E. coli Trp+ reversion test as a plate assay; the E. coli Gal+ forward mutation test; and the Saccharomyces cerevisiae assay for mitotic recombination. Lead chromate is mutagenic in Salmonella and in Saccharomyces and is thus identified as a microbial mutagen by this battery. Metabolic activation by rat liver homogenate (S9) is not required for the mutagenic activity of lead chromate. The most statistically significant, positive result is found with a supplementary assay, the E. coli fluctuation test. To determine whether the lead ion and/or the chromate ion were responsible for the mutagenicity observed, lead chloride and chromium trioxide (chromic acid) were also tested. In E. coli fluctuation test, the ranges of maximal mutagenicity for chromium trioxide and lead chromate overlap at the concentration 10(-5)M, whereas lead chloride shows no mutagenicity and little lethality at concentrations up to 10(-3)M. Thus, it appears that the chromate ion is responsible for the mutagenicity of lead chromate.

Chromates↗

PubMed2005

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.

Bacteria↗

PubMed2003

Distribution of microorganisms in the subsurface of the manus basin hydrothermal vent field in Papua New Guinea.

The distribution of microorganisms in the subsurfaces of hydrothermal vents was investigated by using subvent rock core samples. Microbial cells and ATP were detected from cores taken at depths of less than 99.4 and 44.8 m below the seafloor (mbsf), respectively. Cores from various depths were incubated anaerobically with a heterotrophic medium. Growth at 60 and 90 degrees C was ascribed to a Geobacillus sp. in the 448.6- to 99.4-mbsf cores and a Deinococcus sp. in the 64.8- to 128.9-mbsf cores, respectively, based on the 16S ribosomal DNA analysis.

Anaerobiosis↗

PubMed2000

Detection of emerging resistance patterns within longitudinal surveillance systems: data sensitivity and microbial susceptibility.

Communicating information from antimicrobial resistance surveillance study data to microbiologists and physicians can be challenging. Large amounts of data, commonly reaching millions of MICs or zone 20diameter endpoints, must be analysed and condensed to easily-read tables or figures. Furthermore, data must not be prejudged relative to susceptibility categories, because of the diverse nature of interpretive criteria available internationally. An attempt must be made to present results of all surveillance studies in a mode that can be reinterpreted for immediate use in different geographical areas, or used to compare future data with relative ease and high accuracy. Such data displays require peer-reviewed journals to permit greater numbers of more complex tables to present results. The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) study is a year-on-year global surveillance programme in medical centres where meropenem is available for use. We have developed a presentation strategy that expands the long-term clinical value of MYSTIC results. In addition to statistical parameters, tables of cumulative percentages or numbers of strains inhibited at each tested antimicrobial concentration will be presented. Alternative figures (Finland-o-grams) could also be used, but these generally lack precise extractable rates and require more journal space. Regardless of study design, promotion of this presentation philosophy enhances any surveillance study's value to each reader or user and facilitates application to locally appropriate interpretations. The widespread use of these analysis and presentation principles as benchmarks by various resistance studies and networks is strongly encouraged, particularly by investigations across international boundaries.

Journal Article↗