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PubMed2001

Microbial detection with low molecular weight RNA.

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Biomarkers↗
PubMed2005

Using redox potential to detect microbial activities during clavulanic acid biosynthesis in Streptomyces clavuligerus.

Production of clavulanic acid (CA) by Streptomyces clavuligerus in a shake-flask culture increased from 92 to 180 mg l(-1) with an increased O2 transfer efficiency (0.039 --> 0.058 s(-1)), which maintained the redox potential values above -250 mV. Compared with traditional measures, such as dissolved O2 concentration and respiratory activity, the redox potential can easily be determined and correlates closely with CA production. It can therefore be used to monitor microbial activities during biosyntheses of secondary metabolites.

Bioreactors↗
PubMedDate not supplied

Rapid ribosequencing--an effective diagnostic tool for detecting microbial infection.

BACKGROUND: Rapid and reliable identification of microorganisms is a prerequisite for the diagnosis and subsequent treatment of infectious diseases. The identification of pathogenic bacteria is traditionally based on their isolation from clinical samples and propagation on culture medium in the routine laboratory. However, despite clinical signs of infection, culture of the pathogenic agent often fails. This may be due to a low number of microorganisms, prior antibiotic treatment, nonculturable microorganisms or specific culture requirements for presently unknown pathogens. Amplification and sequencing of the entire prokaryotic 16S-rRNA is time consuming, labor intensive and expensive. MATERIALS AND METHODS: We describe here a procedure for the identification of a wide range of known and unknown clinically relevant microorganisms by sequencing a small, but highly informative region of the prokaryotic 16S-rRNA gene. This rapid ribosequencing method was evaluated with various reference strains and with clinical samples including eye anterior chamber fluid, cerebrospinal fluid (CSF) and blood cultures. RESULTS: All sequences obtained from the reference strains corresponded to the sequences in databases. We correlated severe eye infection with the isolation of Pseudomonas putida, neurological disorder with Tropheryma whippelii and disseminated visceral abscesses in a child with Blastobacter denitrificans. CONCLUSION: We consider the rapid ribosequencing method as a promising new tool for the analysis of infectious agents in primarily sterile body fluids where conventional culturing of microorganisms fails.

Bacteria↗
PubMed2005

Evaluation of the Bactec microbial detection system for culturing miscellaneous sterile body fluids.

This study sought to evaluate the efficacy of the Bactec blood culture system for culturing sterile body fluids, in comparison with traditional culture methods. A total of 906 specimens were cultured using both conventional media and the Bactec detection system. Differences in numbers of isolates determined by each system were compared. Clinically significant microorganisms were isolated from 15.3% (139) of 906 specimens by both the Bactec system and conventional culture, whereas for 8.8% (80) of the specimens a positive culture was obtained by Bactec only. Of the 80 specimens in which growth was detected only in the Bactec blood culture system, 11.0% were comprised of cerebrospinal fluid, 9.9% peritoneal fluid, 4.9% pleural fluid, 4.5% synovial fluid and 9.1% bone marrow materials. Strains of Brucella melitensis, Neisseria meningitidis, Pseudomonas fluorescens and Rothia dentocariosa were cultured only by the Bactec system. In conclusion, the Bactec blood culture system might be advantageous for isolation of fastidious microorganisms such as Brucella, especially from cerebrospinal and peritoneal fluid specimens.

Bacteria↗
PubMed1978

Multichannel electrochemical microbial detection unit.

A compact multichannel unit for electrochemical detection of microorganisms that automatically displays detection time length is described. This unit was successfully tested with various members of the Enterobacteriaceae group.

Bacteriological Techniques↗
PubMed1974

Microbial detection method based on sensing molecular hydrogen.

A simple method for detecting bacteria, based on the time of hydrogen evolution, was developed and tested against various members of the Enterobacteriaceae group. The test system consisted of (i) two electrodes, platinum and a reference electrode, (ii) a buffer amplifier, and (iii) a strip-chart recorder. Hydrogen evolution was measured by an increase in voltage in the negative (cathodic) direction and recorded on a strip-chart recorder. Hydrogen response curves consisted of (i) a lag period, (ii) a period of rapid buildup in potential due to hydrogen, and (iii) a period of decline in potential. A linear relationship was established between inoculum size and the time hydrogen was detected (lag period). Lag times ranged from 1 h for 10(6) cells/ml to 7 h for 10(0) cells/ml. For each 10-fold decrease in inoculum, length of the lag period increased 60 to 70 min. Mean cell concentrations at the time of hydrogen evolution were 10(6)/ml. Based on the linear relationship between inoculum size and lag period, these results indicate the potential application of the hydrogen-sensing method for rapidly detecting coliforms and other gas-producing microorganisms in a variety of clinical, food, and other samples.

Bacteriological Techniques↗
PubMed2002

Evaluation of a plastic nonvented aerobic blood culture bottle for use with the BacT/ALERT microbial detection system.

The current BacT/ALERT SA (BTA SA) aerobic blood culture bottle is made from glass, does not require venting, and contains a liquid emulsion sensor (LES). Its performance has been shown to be equivalent to that of the vented standard aerobic culture bottle. A further-improved version of the BTA SA bottle, designated the BacT/ALERT plastic SA (BTA PSA) culture bottle, is made from clear plastic to prevent breakage, does not require venting, and contains a modified LES (LES 2) to reduce the possibility of false positives. The BTA PSA provides a practical alternative to the current glass version of this bottle. The plastic bottle is also comparable to the current glass bottle in transparency and growth performance and additionally minimizes the exposure to infectious agents due to glass bottle breakage.

Aerobiosis↗
PubMed1977

Preprototype of an automated microbial detection and identification system: a developmental investigation.

The AutoMicrobic System is an automated, computerized instrument that uses highly selective media and an optical system for detection, enumeration, and identification of bacteria and some yeasts in 13 h. A preprototype instrument (AutoMicrobic System-1) and its urine culture kit (Identi-Pak), developed for the detection, enumeration, and identification of eight species or groups of bacteria and of Candida species and Torulopsis glabrata in urine specimens, was evaluated during its development. An overall agreement of approximately 90% between the preprototype instrument and conventional (manual) culture methods has been obtained both with 1,473 seeded (simulated) and 1,688 clinical (mono- or polymicrobial) specimens containing 70,000 (or more) colony-forming units per ml of Escherichia coli, Klebsiella-Enterobacter species, Proteus species, Citrobacter freundii, Serratia species, group D enterococci, or yeasts (Candida species and T. glabrata). Lower agreements in identification were obtained with Pseudomonas aeruginosa-containing (average of 75% in clinical specimens) and Staphylococcus aureus-containing (76%) specimens. Comparison of specimens tested simultaneously in two preprototype systems resulted in =/<4% disagreement; true negativity agreements in all specimen groups tested were at least 94%. Among problems remaining are adaptation of system for specimens other than urine, improvement of sensitivity for P. aeruginosa and S. aureus, and standardization of manual methods used for comparison and validation.

Bacteria↗
PubMed1993

Evaluation of an aseptic technique testing and challenge kit (ATTACK).

Currently, there is considerable discussion and concern about quality assurance when sterile pharmaceuticals are prepared by pharmacists and other health professionals. This study describes the utility of a kit made by Marsam Pharmaceutics Inc. in detecting microbial contamination during simulated drug transfers by syringe. Common microbial detection techniques were incorporated in a simple series of transfers intended to simulate actual use. Growth promotion studies were carried out using Trypticase Soy Broth initially in tubes and then in vials (as supplied in the kit). Three test organisms were employed (Staphylococcus epidermidis, Bacillus subtilis, and Candida albicans) and inoculated at three levels, < 1000, < 100, and < 10 CFU/mL. All studies demonstrated growth occurring in 100% of the trials in 8 days. Similar studies were initiated in stored media (32 month) to determine the effects of time on the ability of the medium to support growth. Growth promoting ability of 32 month old media showed no significant difference. 100% of the vials inoculated showed growth in 8 days. Once the growth promoting qualities of the medium and vials was established a kit was developed then a study to determine the effectiveness of the kit as used was undertaken. Kits were manipulated according to the directions for use by a trained individual, under aseptic conditions and by an untrained individual in an area described as less than desirable. No growth occurred in the vials of the 10 kits used to illustrate good technique and good environmental conditions with 30% (3 of 10) of the kits showing contamination when transfers by syringe were carried out in the less than desirable setting.(ABSTRACT TRUNCATED AT 250 WORDS)

Asepsis↗
PubMed1984

Rapid, inexpensive method for specific detection of microbial beta-lactamases by detection of fluorescent end products.

A rapid method was developed for specific detection of microbial beta-lactamases which uses ampicillin and cephalexin as substrates. The end products (open beta-lactam ring forms) generated after separately incubating either substrate with beta-lactamase-producing organisms initially were separated from the unhydrolyzed substrates by high-voltage electrophoresis at pH 2.1. The end products of both antibiotics were highly fluorescent and could be analyzed visually and semiquantitatively under a long-wave UV lamp. Application of 5 microliters of the same incubation mixture onto filter paper without subsequent electrophoretic separation also resulted in development of fluorescence after brief heating at 120 degrees C for 5 min. This spot test differentiates penicillinase activity from cephalosporinase activity and distinguishes between beta-lactamase and acylase activities, since the end products of acylase [the common side chain, D(-)-alpha-aminophenylacetic acid, and the intact beta-lactam nuclei, 6-aminopenicillanic acid and 7-aminodeacetoxycephalosporanic acid] are not fluorescent. This method was relatively rapid, inexpensive, and more sensitive than the chromogenic cephalosporin (nitrocefin) method when 21 strains of 7 gram-positive species and 77 strains of 29 gram-negative species of bacteria were tested.

Amidohydrolases↗
PubMed1998

The detection of microbial DNA in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients.

OBJECTIVE: The purpose was to determine the sensitivity of detecting microbial DNA in the blood of surgical patients as a measure for diagnosing systemic infection and/or translocation from the gut. SUMMARY BACKGROUND DATA: 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. METHODS: DNA was extracted from the blood of 40 surgical patients and 20 healthy controls. Polymerase chain reaction (PCR) techniques were used to amplify genes from Escherichia coli, Bacteroides fragilis, and a region of 16S ribosomal RNA found in many gram-positive and -negative bacteria. RESULTS: Bacterial DNA genes were not detected in healthy volunteers but were found in all patients with positive blood cultures. All eight transplant patients receiving OKT3 therapy had microbial DNA in their blood, possibly indicating translocation from the gut. Sixty-four percent of critically ill patients had microbial DNA detected in their blood, but only 3 (14%) had positive blood cultures. CONCLUSIONS: The PCR method is more sensitive than blood cultures for detecting bacterial components in the blood of critically ill surgical patients and may detect microbial translocation from the intestine.

Adolescent↗
PubMed1980

Automated detection of microbial growth in blood cultures by using stainless-steel electrodes.

Sterile stainless-steel electrodes implanted in blood culture bottles and monitored electronically were used to detect growth of microorganisms. Each blood culture bottle contained 100 ml of medium and was inoculated with 10 ml of blood seeded with either 300 or 50 colony-forming units of one of several bacterial or yeast species that are commonly isolated from clinical blood cultures. Growth was indicated by a voltage change of at least 0.1 mV/min with an increasing slope over at least three consecutive 15-min intervals. This method was compared to the conventional visual method for detecting microbial growth in broth. Growth detection by both techniques was confirmed by subculture to solid media. Of the 163 cultures seeded with the high inoculum (300 colony-forming units) and confirmed as being positive, 148 (90.8%) were positive by the electronic detection system (EDS). At the lower inoculum (50 colony-forming units), 47 of 53 (88.7%) positive cultures were detected by EDS. Twelve of the 21 false-negatives by the EDS were cultures seeded with Cryptococcus neoformans. Excluding C. neoformans, the rate of detection of growth was 96.0%. Microbial growth was detected an average of 18 h earlier by EDS than by the conventional system in 176 (90.2%) of the cultures. Also examined were 156 patient blood cultures: 13 were positive both by EDS and by conventional methods.

Bacteria↗
PubMed2003

Microarrays for bacterial detection and microbial community analysis.

Several types of microarrays have recently been developed and evaluated for bacterial detection and microbial community analysis. These studies demonstrated that specific, sensitive and quantitative detection could be obtained with both functional gene arrays and community genome arrays. Although single-base mismatch can be differentiated with phylogenetic oligonucleotide arrays, reliable specific detection at the single-base level is still problematic. Microarray-based hybridization approaches are also useful for defining genome diversity and bacterial relatedness. However, more rigorous and systematic assessment and development are needed to realize the full potential of microarrays for microbial detection and community analysis.

Bacteria↗
PubMed1966

Comparative levels and types of microbial contamination detected in industrial clean rooms.

The primary objective of this study was to determine quantitatively and qualitatively the predominant types of microbial contamination occurring in conventional and laminar flow clean rooms. One horizontal laminar flow, three conventional industrial clean rooms, and three open factory areas were selected for microbiological tests. The results showed that as the environment and personnel of a clean room were controlled in a more positive manner with respect to the reduction of particulate contamination, the levels of airborne and surface microbial contaminants were reduced accordingly. The chief sources of microbial contamination were associated with the density and activity of clean room personnel. In addition, the majority of microorganisms isolated from the intramural air by air samplers were those indigenous to humans. Studies on the fallout and accumulation of airborne microorganisms on stainless-steel surfaces showed that, although there were no significant differences in the levels of microbial contamination among the conventional clean rooms, the type of microorganism detected on stainless-steel surfaces was consistently and significantly different. In addition, the "plateau phenomenon" occurred in all environments studied. It was concluded that the stainless-steel strip method for detecting microbial accumulation on surfaces is efficient and sensitive in ultra-clean environments and is the most reliable and practical method for monitoring microbial contamination in future class 100 clean rooms to be used for the assembly of spacecraft which will be sterilized.

Air Microbiology↗
PubMedDate not supplied

A method for the rapid detection of microbial contaminants in animal cell culture processes.

The early detection of microbial contamination is an important issue in the production of biopharmaceuticals using animal cell culture systems. A new method, based on the ChemScan RDI analyser, was evaluated for the rapid detection and enumeration of low concentrations of microorganisms within a large population of animal cells and in cell culture media. The method was tested with suspension cell cultures and is applicable to adherent cell cultures. In both cases, the method uses an initial step to eliminate the animal cells followed by collection and fluorescent labelling of the viable microbial contaminants on a filter membrane. Total counts of the viable microorganisms were obtained after analysis of the membrane by the ChemScan RDI analyser. The results showed that the ChemScan RDI detected individual bacterial cells after filtration of the pre-treated animal cell culture. The detection limit of the ChemScan RDI was less than 10 bacteria/ml in cell culture containing 10(6) mammalian cells/ml and one bacterium in 500 ml of cell culture medium. A strong correlation between the standard plate count and the ChemScan RDI was observed, even at low bacterial concentrations. The total time for each analysis was less than two hours.

Animals↗
PubMed2003

Possibilities for the detection of microbial life on extrasolar planets.

We consider possibilities for the remote detection of microbial life on extrasolar planets. The Darwin/Terrestrial Planet Finder (TPF) telescope concepts for observations of terrestrial planets focus on indirect searches for life through the detection of atmospheric gases related to life processes. Direct detection of extraterrestrial life may also be possible through well-designed searches for microbial life forms. Satellites in Earth orbit routinely monitor colonies of terrestrial algae in oceans and lakes by analysis of reflected ocean light in the visible region of the spectrum. These remote sensing techniques suggest strategies for extrasolar searches for signatures of chlorophylls and related photosynthetic compounds associated with life. However, identification of such life-related compounds on extrasolar planets would require observations through strong, interfering absorptions and scattering radiances from the remote atmospheres and landmasses. Techniques for removal of interfering radiances have been extensively developed for remote sensing from Earth orbit. Comparable techniques would have to be developed for extrasolar planet observations also, but doing so would be challenging for a remote planet. Darwin/TPF coronagraph concepts operating in the visible seem to be best suited for searches for extrasolar microbial life forms with instruments that can be projected for the 2010-2020 decades, although resolution and signal-to-noise ratio constraints severely limit detection possibilities on terrestrial-type planets. The generation of telescopes with large apertures and extremely high spatial resolutions that will follow Darwin/TPF could offer striking possibilities for the direct detection of extrasolar microbial life.

Biological Evolution↗