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PubMed2006

Evaluation of the sterility test for detection of microbial contaminants of allografts.

Tissue banks routinely use a sterility test to determine the suitability of processed tissue prior to release. However, many tissue banks also accompany the sterility test with additional types of media to ensure detection of slower-growing or more fastidious organisms that may not be detected in the standard two media/two incubation temperature sterility test. This study was designed to determine if a standard sterility test is capable of detecting the wide variety of organisms that may be isolated from human tissue thereby making the additional plated media unnecessary. More than 100 isolates, representing more than 90 different species were tested. All isolates exhibited growth in at least one of the two standard sterility test media within the 14-day incubation period.

Fungi↗

PubMedDate not supplied

Enzyme immunoassays for the detection of microbial antigens and prospects for improved assays.

The rapid diagnosis of viral infections is an important tool in the management of patients with infectious diseases. Solid-phase enzyme immunoassays have proved to be useful tools for the direct detection of the antigens of some viruses directly in clinical specimens. Such assays have been particularly useful in the diagnosis of viral infections in the gastrointestinal and respiratory tracts. However, standard solid-phase enzyme immunoassays often do not display sufficient sensitivity for the diagnosis of all cases of viral infections. Techniques which might be utilized to increase the sensitivity of solid-phase immunoassays include the use of monoclonal antibodies to maximize the efficiency of the antigen-antibody interactions and the use of high-turnover enzymes to increase the amount of signal generated by the ensuing enzyme-substrate reactions. In addition, techniques making use of nucleic acid hybridization have a great deal of potential for the accurate detection of viral nucleic acids in human body fluids. The successful application of these techniques to the diagnosis of viral infections could lead to a marked improvement in the care of patients with suspected infectious diseases as well as to a decrease in the transmission of viral infections to high-risk individuals.

Affinity Labels↗

PubMed1976

Lecithin agar for detection of microbial phospholipases.

Lecithin agar was developed on which phospholipase C produced turbid zones and phospholipase A produced clear zones. Reactions on lecithin agar agreed 74% of the time with reactions in egg yolk broth. On lecithin agar, interpretation was easier, phospholipase A was detectable, and opaque zones were visible 1 or 2 days earlier than on egg yolk agar. All constituents of the medium can be autoclaved.

Agar↗

PubMed2002

Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo-Roche Rapid Anthrax Test.

Rapid-cycle real-time polymerase chain reaction has immediate and important implications for diagnostic testing in the clinical microbiology laboratory. In our experience this novel testing method has outstanding performance characteristics. The sensitivities for detecting microorganisms frequently exceed standard culture-based assays, and the time required to complete the assays is considerably shorter than that required for culture-based assays. We describe the principle of real-time polymerase chain reaction and present clinical applications, including the detection of Bacillus anthracis, the causative agent of anthrax. This latter test is commercially available as the result of a collaborative venture between Mayo Clinic and Roche Applied Science, hence the designation The Mayo-Roche Rapid Anthrax Test.

Anthrax↗

PubMed2005

Contemporary methods for detection of microbial infections in transplanted tissues.

Infective processes developing after transplantation are diagnosed mainly in the specimens harvested from blood and exudates. Not much attention has been focused on identification of microorganisms present in the graft or penetrating the graft from the recipient. Although a progress has been made with respect to detection of viruses there is still little knowledge on the pathological role of bacteria, transplanted together with the graft or acquired from the recipient, in evoking or enhancing the rejection reaction (heterologous immune response). Recently developing molecular techniques allow us to detect bacterial genetic materials in clinical specimens. This article reviews commonly used methods for detection, identification and typing bacterial pathogens. It focuses on specific and universal methods based on gene sequencing, PCR and techniques based on polymorphic DNA. We also present the examples of genes and microorganisms analysed in laboratories. Together with conventional microbiological methods the molecular tests can serve as a key for better understanding the bacterial presence in tissues, not necessarily causing inflammation, quite often being commensal, but also evoking host reaction under certain so far not defined circumstances.

Bacterial Infections↗

PubMedDate not supplied

Genetic methods for the detection of microbial pathogens. Identification of enterotoxigenic Escherichia coli by DNA colony hybridization: collaborative study.

Enteropathogenic Escherichia coli strains may produce a cholera-like, heat-labile enterotoxin (LT) as a virulence factor. The gene that codes for LT can be purified by recombinant DNA techniques and used as a genetic probe for DNA hybridization. These probes detect enterotoxigenic strains as well as strains that may not manifest toxin production but carry the genetic information to do so. In this study, 13 laboratories tested 3 known and 25 unknown (10 positive and 15 negative) cultures of E. coli for the presence of the LT gene. The isolates had been tested and classified by the mouse Y-1 adrenal cell test and an enzyme-linked immunosorbent assay. Cultures were spotted on nitrocellulose filters on MacConkey agar and incubated. Colonies were lysed in situ and their DNA was hybridized to 32P-labeled, purified LT gene DNA (provided to the collaborators). Positive colonies were identified by autoradiography. Of 325 samples, 315 (96.9%) were identified correctly and 10 were misclassified; there were 6 false negative and 4 false positive identifications. Chi-square values indicated that the method agreed with the previous classification and was equally efficient in distinguishing positive and negative samples (95.7 and 98.1%, respectively). The method has been adopted official first action.

Autoradiography↗

PubMed2002

Microbial complexes detected in the second/third molar region in patients with asymptomatic third molars.

PURPOSE: Our goal was to report the detection and levels of pathogenic bacteria in subgingival plaque samples taken from the distal of all second molars in 295 patients with asymptomatic third molars. PATIENTS AND METHODS: Data assessing oral health were collected from each of these healthy patients (ASA Classes I and II). Probing depth (PD), at 6 sites per tooth, including third molars, was obtained to determine periodontal status. Subgingival plaque samples were taken from the distal of all second molars before periodontal probing. The presence and levels of 11 bacterial species were determined using whole chromosomal DNA probes and checkerboard DNA-DNA hybridization. Detected bacterial species were grouped into clusters of periodontal pathogens designated as "red" or "orange" complex microorganisms as described by Socransky et al (J Clin Periodontal 25:134, 1998) who found an association of these specific microorganisms with periodontitis. RESULTS: As a group these relatively young patients were periodontally healthy. "Orange and red" complex microorganisms were detected at levels equal to or greater than 10(5) more often if patients had a PD equal to or greater than 5 mm with periodontal attachment loss at the distal of second molars or around third molars at their entry examination. In patients with no PD equal to or greater than 5 mm in the third molar region, "orange and red" complex microorganisms were detected at levels equal to or greater than 10(5) more frequently than would be anticipated in patients with little clinical evidence of periodontal disease. CONCLUSIONS: The clinical findings of increased periodontal PDs and periodontal attachment loss coupled with colonization of periodontal pathogens support the concept that clinical and microbial changes associated with the initiation of periodontitis may present first in the third molar region in young adults.

Adolescent↗

PubMed1998

Evaluation of PCR assays in presence of antibody to thermostable DNA polymerases for detection of microbial agents: avoiding false negative results for specimen containing low-titer agent.

The serial low-titer specimens of Influenza A virus and Adeno virus type 7 were tested for the presence of virus specific genes by PCR based on Tth DNA polymerase and by that based on Taq DNA polymerase, in the absence and presence of antibody to the respective DNA polymerases. Increased product DNA synthesis and higher sensitivity of detection were observed in the presence of antibody compared to those in the absence of antibody. 10- to 100- fold lower titer specimen of Influenza A virus and 10-fold lower titer specimen of Adeno virus could be detected in the presence of antibody than those detected in the absence of antibody to the appropriate DNA polymerase, in a PCR.

Adenoviruses, Human↗

PubMed2001

Confocal microscopy and microbial viability detection for food research.

Confocal microscopy offers several advantages over other conventional microscopic techniques as a tool for studying the interaction of bacteria with food and the role of food microstructure in product quality and safety. When using confocal microscopy, samples can be observed without extensive preparation processes, which allows for the evaluation of food without introducing artifacts. In addition, observations can be made in three dimensions without physically sectioning the specimen. The confocal microscope can be used to follow changes over a period of time, such as the development of the food structure or changes in microbial population during a process. Microbial attachment to and detachment from food and food contact surfaces with complex three-dimensional (3-D) structures can be observed in situ. The fate of microbial populations in food system depends on processing, distribution, and storage conditions as well as decontamination procedures that are applied to inactivate and remove them. The ability to determine the physiological status of microorganisms without disrupting their physical relationship with a food system can be useful for determining the means by which microorganisms survive decontamination treatments. Conventional culturing techniques can detect viable cells; however, these techniques lack the ability to locate viable cells in respect to the microscopic structures of food. Various microscopic methods take advantage of physiological changes in bacterial cells that are associated with the viability to assess the physiologic status of individual cells while retaining the ability to locate the cell within a food tissue system. This paper reviews the application of confocal microscopy in food research and direct observation of viable bacteria with emphasis on their use in food microbiology.

Bacteria↗

PubMed2002

Quantitative detection of microbial genes by using DNA microarrays.

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.

Dioxygenases↗

PubMed1977

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.

Bacteria↗

PubMed1998

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil.

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent-bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR.

Journal Article↗

PubMed1991

[Detection of microbial protein synthesis in the small intestine of sheep using an intraduodenal 15N-urea infusion].

Sheep (3 animals, 50 kg LW) with reentrant cannulas in duodenum and at the end of the ileum received 700 g hay and 800 g alfalfa pellets per animal and day. In a previous 1st period of three days duodenal digesta and in a 2nd period of four days ileal digesta were collected and stored deep frozen. In the main period the digesta flow was interrupted for 28 hours. The duodenal and ileal digesta were collected quantitatively. The previously collected duodenal and ileal digesta portions were introduced hourly. The duodenal digesta was supplemented with 15N-labelled urea for a 24 hour period. 4.5% of the introduced 15N-excess were detected at the end of the ileum in the 24 hour period. 5.6% of the 15N-excess at the end of the ileum were incorporated in bacterial protein. It was measured that the ileal digesta contained 4.62 g N in the TCE precipitable fraction and 24.4% of the TCE precipitable N-fraction was bacterial nitrogen.

Animals↗

PubMed1994

[Serologic detection of microbial diseases--present status and perspectives].

The cost of serological diagnostic products amounts to more than a quarter of total laboratory material expenses. The major problem in serological diagnostics is correct interpretation of the presence of specific antibodies. Aspects influencing diagnostic conclusions drawn from serological finding include: 1. Features of infectious agents and pathogenesis of infection. 2. Principle of serological assay. 3. Condition of antigen used in the assay. 4. Individual reactivity of the patient. 5. Temporal data and data on the course of the disease. Modern serological methods such as ELISA and indirect immunofluorescence have, compared to classical techniques (i.e. complement fixation test, agglutination, immunoprecipitation and neutralization), a number of advantages. Nevertheless, they also have drawbacks of and classical methods should be used at least in selected cases for a number of years in future. Novel approaches such as immunoblotting, body fluid antibody detection, IgA and IgE determination and low avidity antibody detection are gaining importance and promise new insights.

Humans↗

PubMed2001

Detection of microbial volatile organic compounds (MVOCs) produced by moulds on various materials.

Twelve fungal species were screened for microbial volatile organic compounds (MVOCs): Aspergillus fumigatus, A. versicolor, A. niger, A. ochraceus, Trichoderma harzianum, T. pseudokoningii, Penicillium brevicompactum, P. chrysogenum, P. claviforme, P. expansum, Fusarium solani and Mucor sp. More than 150 volatile substances derived from fungal cultures have been analysed by head-space solid-phase microextraction (HS-SPME). Each species had a defined MVOC profile which may be subjected to considerable modification in response to external factors such as cultivation on different substrata. The cultivation on different substrata changes the number and concentration of MVOCs. Species-specific volatiles may serve as marker compounds for the selective detection of fungal species in indoor environments. Examination of MVOCs from indoor air samples may become an important method in indoor air hygiene for the detection of type and intensity of masked contamination by moulds.

Air Pollution, Indoor↗

PubMed2005

Detection of microbial DNA in the blood of surgical patients for diagnosing bacterial translocation.

Bacterial translocation sometimes occurs in patients during surgical stress and is associated with an increased incidence of septic morbidity. However, no reliable method has been established for diagnosing bacterial translocation in humans. Identification of minute quantities of microbial-specific DNA has been made possible using polymerase chain reaction (PCR) techniques. The aims of this study were to determine the prevalence of bacterial translocation in patients with surgical stress using PCR techniques and to evaluate the usefulness of blood PCR techniques for diagnosing bacterial translocation. DNA was extracted from the blood of 52 surgical patients (24 elective major surgery patients and 28 septic patients) and 10 healthy controls. PCR techniques were used to amplify genes from Escherichia coli, Bacteroides fragilis, a region of 16S ribosomal RNA found in many gram-positive and gram-negative bacteria, and Candida albicans. Bacterial and Candida albicans DNA were not detected in healthy volunteers. Enteric bacterial DNA was detected in patients with hepatic lobectomy, and Candida albicans DNA was detected in patients with esophagectomy on the first postoperative day. Enteric bacterial and Candida albicans DNA were detected in septic patients with findings diagnostic of bacterial translocation, such as small bowel obstruction, ulcerative colitis, or supramesenteric arterial occlusion or in those who had undergone chemotherapy for advanced colon cancer. However, none of the patients were positive by the blood culture technique. The PCR method is more sensitive than blood cultures for detecting bacterial components in the blood of septic patients and is a valuable tool for verifying bacterial translocation in patients who have undergone hepatic lobectomy or esophagectomy. It is also valuable in septic patients who do not have a defined focus of infection.

Bacterial Translocation↗

PubMed2002

Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1.

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 [4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate], in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.

Bacteria↗

PubMed2025

Comparative performance of sponge versus flocked swabs for culture-based and metagenomic detection of microbial contamination in the healthcare environment.

BACKGROUND: Identifying optimal methods for sampling surfaces in the healthcare environment is critical for future research requiring the identification of multidrug-resistant organisms (MDROs) on surfaces. METHODS: We compared 2 swabbing methods, use of a flocked swab versus a sponge-stick, for recovery of MDROs by both culture and recovery of bacterial DNA via quantitative 16S polymerase chain reaction (PCR). This comparison was conducted by assessing swab performance in a longitudinal survey of MDRO contamination in hospital rooms. Additionally, a laboratory-prepared surface was also used to compare the recovery of each swab type with a matching surface area. RESULTS: Sponge-sticks were superior to flocked swabs for culture-based recovery of MDROs, with a sensitivity of 80% compared to 58%. Similarly, sponge-sticks demonstrated greater recovery of Staphylococcus aureus from laboratory-prepared surfaces, although the performance of flocked swabs improved when premoistened. In contrast, recovery of bacterial DNA via quantitative 16S PCR was greater with flocked swabs by an average of 3 log copies per specimen. CONCLUSIONS: The optimal swabbing method of environmental surfaces differs by method of analysis. Sponge-sticks were superior to flocked swabs for culture-based detection of bacteria but inferior for recovery of bacterial DNA.

Humans↗