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PubMed1982

[Comparative study with 2 new and 8 known nutrient media for cultivation of fastidious and nonfastidious microbial agents from cerebrospinal fluid and other body fluids].

Rapid physical, biochemical and immunological methods may be useful in the detection of microbial agents in cerebrospinal fluid and in other body fluids. However, these methods are no substitution for the cultivation of the microbial agents. Microorganisms which are most frequently responsible for meningitis are fastidious in their growth requirements. Their detection with the help of conventional blood culture media which are not supplemented with blood or its components, leads to a high quota of false-negative results. Taking this problem into consideration, the authors developed the following two new media: "MOPS Electrolyte Broth A" for culturing obligate aerobic and facultative anerobic microorganisms, and "MOPS Electrolyte Broth AN" for culturing facultative anaerobic and obligate anaerobic bacteria. Performance tests have been carried out with the two above mentioned media and eight commercially manufactured blood culture media in original bottles. Twenty representative test strains including the most important and fastidious microbial agents of meningitis have been considered in this study. The inoculum size was about 10(2) CFU per culture bottle. The two new media, which were not supplemented with blood or body fluids, proved to be more effective than the conventional blood culture media supplemented with 10% fresh human blood for culturing the considered spectrum of microorganisms.

Aerobiosis↗
PubMedDate not supplied

Molecular characterization of anaerobic microbial communities from benzene-degrading sediments under methanogenic conditions.

Anaerobic benzene degradation was confirmed in microbial communities enriched from Baltimore Harbor (Baltimore, MD) sediments under methanogenic conditions. Molecular characterization based on 16S rDNA gene sequences revealed that the strains in the communities were diversely affiliated with such phylogenetic branches as the Bacteroidetes, Euryarchaeota, Firmicutes, and Thermotogae phyla. Of interest was that the majority of the microbial populations detected in these cultures were closely related to the members of dechlorinating microbial communities. Further, some of those species were previously found in naphthalene- or phenanthrene-degrading methanogenic communities. Finally, this result could be used to design targeted isolation strategies for anaerobic benzene-degrading strains under methanogenic conditions.

Bacteria, Anaerobic↗
PubMed2006

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens.

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.

Bacteria↗
PubMedDate not supplied

[Research of the soluble microbial substances in organic fluids for the rapid diagnosis of some infections and particularly of bacterial meningitis (author's transl)].

A number of immunological and non-immunological techniques have been recently used to detect soluble microbial substances in body fluids of patients with acute meningitis, bacteremia, and lobar pneumonia. By the immunological methods capsular highly polymerized polisaccharide group- or type-specific antigens of the most common C. N. S. pathogens (N. meningitidis A, B, and C; Str. pneumoniae, H. influenzae type b, E. coli K1, mucoid Pseudomonas, Cryptococcus neoformans) can be detected and quantitated in spinal fluids, sera, urine and other fluids specimens from meningitic patients. Capsular type-specific antigens from pneumococcus, and likely from H. influenzae as well, can be detected in sputum from patients with lower respiratory infection. Among the various techniques, the radioimmunoassay appears as the most sensitive one, but high diagnostic sensitivity can be also achieved by using the latex agglutination, haemoagglutination inhibition and coagglutination tests. Counterimmunoelectrophoresis, however, is still the far most used technique for determining soluble microbial antigens, albeit its sensitivity is significantly less than the one of the above mentioned methods. High specificity and some advantages in serotyping the causal organisms are probably the main reasons of such preferential employment. Among the non-immunological techniques the evaluation of lactate and lactic dehydrogenase has been used by some Author for differentiating between bacterial and non bacterial meningitis, and the limulus test for detecting Gram-negative bacterial endotoxins with a high degree of sensitivity and specificity. Finally, the liquid gas chromatography has been evaluated in detection of some organic products (microbial?), such as acids, amines, neutral compounds, in spinal fluid, allowing the differential diagnosis between bacterial, tuberculous, viral, and cryptococcal meningitis. In the present review sensitivity, specificity, and other properties of each test alone and in comparison with the conventional microbiological methods (Gram and culture) are evaluated and the biological and pathogenic role and significance of the soluble microbial antigens and endotoxin are discussed.

Agglutination Tests↗
PubMed1989

Hybridization of DNA probes with whole-community genome for detection of genes that encode microbial responses to pollutants: mer genes and Hg2+ resistance.

Nucleic acids extracted from microbial biomass without prior culturing were hybridized with probes representing four mer operons to detect genes encoding adaptation to Hg2+ in whole-community genomes. A 29-fold enrichment in sequences similar to the mer genes of transposon Tn501 occurred during adaptation in a freshwater community. In an estuarine community, all four mer genes were only slightly enriched (by three- to fivefold), suggesting that additional, yet uncharacterized, mer genes encoded adaptation to Hg2+.

Adaptation, Physiological↗
PubMed2004

Atmospheric pressure chemical ionization and atmospheric pressure photoionization for simultaneous mass spectrometric analysis of microbial respiratory ubiquinones and menaquinones.

An atmospheric pressure photoionization (APPI) source and an atmospheric pressure chemical ionization (APCI) source were compared for the selective detection of microbial respiratory ubiquinone and menaquinone isoprenologues using tandem mass spectrometry. Ionization source- and compound mass-dependent parameters were optimized individually for both sources, using the available quinone standards. Detection levels for the two ion sources were determined with ubiquinone-6 (UQ6) and menaquinone-4 (MK4, vitamin K2) standards using flow injection analysis and selected reaction monitoring (SRM). With APPI the calculated lower limit of detection (LLOD) was 1.7 fmol microl(-1) for UQ6 and 2.2 fmol microl(-1) for MK4 at a signal-to-noise ratio of 3. These LLODs were at least three times lower than with APCI. The selectivity of detection afforded by SRM detection reduced complex mixture analysis to 3 min per sample by eliminating the need for chromatographic separations. The detection method was successfully applied to quinone quantification in a variety of environmental samples and cell cultures. Adequate amounts of respiratory quinones can be extracted and quantified from samples containing as low as 2 x 10(7) cells.

Atmospheric Pressure↗
PubMed2005

Biomineralization of endolithic microbes in rocks from the McMurdo Dry Valleys of Antarctica: implications for microbial fossil formation and their detection.

In some zones of Antarctica's cold and dry desert, the extinction of cryptoendolithic microorganisms leaves behind inorganic traces of microbial life. In this paper, we examine the transition from live microorganisms, through their decay, to microbial fossils using in situ microscopy (transmission electron microscopy, scanning electron microscopy in back-scattered electron mode) and microanalytical (energy dispersive X-ray spectroscopy) techniques. Our results demonstrate that, after their death, endolithic microorganisms inhabiting Commonwealth Glacier sandstone from the Antarctica McMurdo Dry Valleys become mineralized. In some cases, epicellular deposition of minerals and/or simply filling up of empty moulds by minerals leads to the formation of cell-shaped structures that may be considered biomarkers. The continuous deposition of allochthonous clay minerals and sulfate-rich salts fills the sandstone pores. This process can give rise to microbial fossils with distinguishable cell wall structures. Often, fossilized cell interiors were of a different chemical composition to the mineralized cell walls. We propose that the microbial fossil formation observed was induced by mineral precipitation resulting from inorganic processes occurring after the death of cryptoendolithic microorganisms. Nevertheless, it must have been the organic template that provoked the diffusion of mineral elements and gave rise to their characteristic distribution pattern inside the fossilized cells.

Antarctic Regions↗
PubMed2005

[Early diagnosis of colorectal anastomotic leakages by detection of bacterial genome].

BACKGROUND: Microbial infections and translocation of intestinal bacteria are thought to contribute to multiple system organ failure, but bacterial cultures are often negative in patients with this complication. The purpose of this study was to determine the sensitivity of PCR for detecting microbial DNA in the blood of animals after conducting an experimental model of anastomotic leakage. METHODS: Fourty rats were divided into three groups as follows: Control Group; simple laparotomy group, Anastomosis Group; colon resection and anastomosis group, and Leakage Group; group with colon resection and an anastomosis leaving a 5 mm opening. Blood was drawn form rats before the procedure, and postoperative 3rd and 6th days. DNAs were extracted from these samples and PCR techniques were used to amplify genes of Escherichia coli. Statistical analysis for the percentage of rats with microbial DNA in the blood for all groups was done by Fisher's exact chi- square test. The difference among groups was considered significant if the P value was less than 0.05. RESULTS: Most of the detected Escherichia coli genes were from the Leakage Group, and the detection rate was significant compared to other groups. CONCLUSION: We suggest that PCR could be a useful adjunct tool for immediate diagnosis of anastomotic leakages.

Anastomosis, Surgical↗
PubMed1995

Clinical evaluation of the Vital system compared with the Hémoline diphasic method for the detection of aerobic blood cultures.

In a collaborative study at two university hospitals, the recovery of microorganisms and the speed of detection of microbial aerobic growth by the Vital system (bioMérieux) and a diphasic conventional blood culture system were compared. The Vital system monitors each blood culture bottle every 15 minutes to detect inactivation of fluorescent suspended in the broth medium due to microbial growth. A total of 1086 comparisons were made between the two systems, yielding a total of 117 isolates. Microorganisms were recovered more often from the Vital aerobic bottles (p < 0.05). The Vital system detected 43% of the microorganisms within the first 12 hours of incubation whereas in the same time the conventional system detected only 5% of the microorganisms. The results demonstrate that the Vital system is a reliable, continuous monitoring, fully automated system and an attractive alternative to conventional blood culture methods.

Bacteremia↗
PubMed1991

Operator-induced contamination in cell culture systems.

Operator-induced biological contamination in cell cultures is a multifaceted problem involving the unexpected introduction of other animal cells, microbial and viral contaminants. Detailed studies on animal cell cross contaminations have been performed and published. The frequency of detection of problem cultures has been as high as 36% for one service performed in the USA, with interspecific cross contamination accounting for 25% and human intraspecific contamination representing 11%. Awareness of the potential of this problem plus the application of several characterizations are key factors for its control. For example, fluorescent antibody staining, isoenzyme analyses, cytogenetic evaluations and DNA fingerprinting using molecular probes are needed for quality assurance on master seed stocks. Detection of microbial contamination is relatively straightforward, but the prevalence of mycoplasmal infections in cell cultures used in general research is still a significant problem. Detection services report frequencies of infection varying from 10% upwards, depending upon the country and laboratory of origin. The utilization of prescreened reagents and antibiotic-free cultivation, plus the application of improved procedures, such as fluorescent dyes and molecular probes for detection, provide effective means of avoiding mycoplasma infection and facilitating control. For many viruses, the presence of mycoplasma reduces immunoreactivity, suppresses transcriptase and other enzyme activities, reverses viral neutralization etc. The introduction of viral contaminants into cell cultures is perhaps the most problematic, especially where no cytopathic effect is produced. Few cases are documented where technicians infected with specific viruses have introduced these unwittingly into cultures in their care. The potential exists, however, as reports have appeared documenting the considerable stability of rhinoviruses, respiratory syncytial virus, rotaviruses and others, in aerosols on workers' hands and safety hood surfaces. The infection of cell cultures via other contaminated cells or reagents such as sera is a related problem. In this regard, the infection of transplantable tumor cell lines with lymphocytic choriomeningitis virus from host animals led to an outbreak of the disease in medical center personnel. Similar infection of rat cell lines exposed to animals harboring hantaviruses has been reported. Technical staff in US government laboratories have been infected with human immunodeficiency virus produced in cultured cells. Such serious public health hazards warrant repeated emphasis. The use of multiple cell lines in a given laboratory, including cultures known to be virally infected, compounds the problems and necessitates application of preventive methods both to avoid cross-infections and to document freedom from contamination.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Detection of divergent genes in microbial aCGH experiments.

BACKGROUND: Array-based comparative genome hybridization (aCGH) is a tool for rapid comparison of genomes from different bacterial strains. The purpose of such analysis is to detect highly divergent or absent genes in a sample strain compared to an index strain. Development of methods for analyzing aCGH data has primarily focused on copy number abberations in cancer research. In microbial aCGH analyses, genes are typically ranked by log-ratios, and classification into divergent or present is done by choosing a cutoff log-ratio, either manually or by statistics calculated from the log-ratio distribution. As experimental settings vary considerably, it is not possible to develop a classical discriminant or statistical learning approach. METHODS: We introduce a more efficient method for analyzing microbial aCGH data using a finite mixture model and a data rotation scheme. Using the average posterior probabilities from the model fitted to log-ratios before and after rotation, we get a score for each gene, and demonstrate its advantages for ranking and detecting divergent genes with enlarged specificity and sensitivity. RESULTS: The procedure is tested and compared to other approaches on simulated data sets, as well as on four experimental validation data sets for aCGH analysis on fully sequenced strains of Staphylococcus aureus and Streptococcus pneumoniae. CONCLUSION: When tested on simulated data as well as on four different experimental validation data sets from experiments with only fully sequenced strains, our procedure out-competes the standard procedures of using a simple log-ratio cutoff for classification into present and divergent genes.

Computational Biology↗
PubMed2005

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA.

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

Base Sequence↗
PubMed2000

Colorimetric indicators of microbial contamination in corneal preservation medium.

PURPOSE: To compare acid-base and oxidation-reduction indicators and to investigate the effect of buffer and temperature on the colorimetric detection of microbial growth in corneal preservation media. METHODS: Corneal preservation media containing gentamicin, without or with HEPES buffer, were prepared with either phenol red or AlamarBlue indicators (AccuMed International, Westlake, OH, U.S.A.). Both media were inoculated with Staphylococcus aureus, Streptococcus sanguis, Pseudomonas aeruginosa, Serratia marcescens, or Candida albicans and then incubated at 4 degrees C, 22 degrees C, or 35 degrees C. The pH or percent reduction were determined hourly for eight hours, then daily for one week. RESULTS: The length of time before a confirmed change in pH or reduction occurred varied by microorganism, storage temperature, and buffering capacity. At 4 degrees C, none of the microorganisms caused a detectable pH change in buffered medium within one day after inoculation, although two bacterial species reduced AlamarBlue within four hours. At 22 degrees C and 35 degrees C, all bacteria except P. aeruginosa produced a pH shift within a few hours, and all tested bacterial species reduced AlamarBlue. For bacteria producing detectable pH changes, HEPES-buffered medium took longer to change than medium without HEPES. C. albicans was not detectable in HEPES-buffered medium at any temperature by phenol red and was only detectable by AlamarBlue after 2-3 days at 22 degrees C and 35 degrees C. CONCLUSION: Acidic shifts in refrigerated corneal preservation medium do not occur during contamination by several microorganisms. AlamarBlue, a redox indicator, is more sensitive than phenol red in detecting some bacteria. C. albicans is not reliably detected by pH or redox indicators.

Bacteria↗
PubMed2006

Taxa-area relationships for microbes: the unsampled and the unseen.

The recent observation of a power-law relationship, S proportional A(z), between number of taxa, S, and area, A, for microbial eukaryotes and bacteria suggests that this is one of the few generic relationships in ecology, applicable to plants, animals and microbes. However, the rate of increase in the number of species with area varies from approximately the fourth (z = 0.26) to as little as the 50th root (z = 0.0019) in microbes. This is an enormous range for which no quantitative explanation has been proffered. We show by sampling from synthetic populations that the disparity between sample and community sizes in microbial community surveys means z can be considerably underestimated and accrual of rare taxa with increasing area will not be detectable. Significant microbial taxa-area relationships will only be observed when changes in community structure within samples correlate with area. Thus, the very low z values observed recently cannot be used as the sole evidence in support of any particular community theory of community assembly. More generally, this suggests that our search for patterns and laws in the microbial world will be profoundly influenced and, potentially distorted by the sample sizes that are typical of microbial community surveys.

Bacteria↗
PubMed1999

Culture shock. Molecular methods for diagnosis of infectious diseases.

Nucleic acid detection methods, such as polymerase chain reaction (PCR), can often detect specific microbial pathogens, virulence markers and antimicrobial resistance genes more rapidly and with greater sensitivity and specificity than culture and conventional identification and susceptibility testing. Multiplex PCR can detect multiple genes in a single assay; this capability will be greatly extended by new techniques such as the DNA chip. However, limitations and pitfalls of nucleic detection methods remain.

DNA Primers↗
PubMed1997

Diet and carcinogen alter the fecal microbial populations of rats.

An analysis of viable bacterial populations enumerated on carbohydrate selective media was used to simulate the colonic environment in vitro and determine if differential media could detect significant microbial shifts due to dietary fiber source, dietary fat source, and carcinogen. Male Sprague-Dawley rats were provided with either pectin or cellulose as a fiber source, either corn or fish oil as a source of fatty acids, and injected with either azoxymethane (AOM), a gastrointestinal carcinogen, or saline in a 2 x 2 x 2 factorial design. At 6 and 10 mo of age, fresh feces were collected, homogenized in anaerobic buffer and anaerobically plated onto differential media. Diets containing pectin supported more anaerobes at 6 mo of age (P < 0.01) than diets containing cellulose. Rats injected with AOM and consuming either pectin or corn oil supported more anaerobes at 10 mo of age (P < 0.05) than rats injected with saline and consuming the same diets. Rats consuming cellulose and receiving AOM but not expressing tumors possessed larger anaerobic populations at 10 mo of age (P < 0.05) than rats consuming cellulose, injected with AOM and expressing tumors. These effects show that gastrointestinal bacterial populations, as measured by carbohydrate specific media, respond to dietary changes such as dietary fiber source, and thus may play a key role in the etiology of colon cancer.

Aging↗
PubMed1997

[Molecular pathologic detection of mycobacteria].

In recent years, significant advances in the diagnosis of mycobacterial infections have been made by the introduction of direct pathogen detection methods. These techniques are usually based on the polymerase chain reaction (PCR) or on a transcription-mediated amplification (TMA) process. The majority of the protocols have been optimized for the detection of mycobacterial nucleic acids in fresh fluid or fresh tissue specimen. Unfortunately pathologists are frequently confronted with the problem that tissues with histologically suspicious lesions have been entirely fixed in formalin. As a result of this routine fixation, DNA and RNA are heavily degraded and the usually high sensitivity of the amplification techniques is greatly impaired. Consequently, only PCR protocols designed for small amplification targets are still suitable for an efficient detection of microbial DNA in formalin-fixed and paraffin-embedded tissues. We therefore adapted PCR assays with amplification products < 200 bp for the detection of M. tuberculosis-complex DNA (targets: IS6110 and 65 kDa-antigen gene) in routine biopsies. Although the sensitivities of the two assays varied significantly with the degree of DNA degradation, we were able to detect M. tuberculosis-complex specific DNA in about 25% of the tissues with a granulomatous inflammation and negative Ziehl-Neelson stain. Recently, we have added a third PCR-assay, which in combination with direct sequencing also allows us to detect DNA from M. leprae and several atypical mycobacteria species. PCR-analysis has significantly improved the diagnosis of mycobacterial infections by supplementing conventional histological examination of formalin-fixed and paraffin-embedded tissues.

DNA, Bacterial↗