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PubMed1992

Controlled comparison of the BacT/Alert and BACTEC 660/730 nonradiometric blood culture systems.

In a collaborative study at three university hospitals, the recovery of microorganisms and the speed of detection of microbial growth by the BacT/Alert (Organon Teknika Corporation, Durham, N.C.) and BACTEC 660/730 (Becton-Dickinson Diagnostic Instrument Systems, Sparks, Md.) nonradiometric blood culture systems were compared. A total of 5,918 comparisons were made between BacT/Alert aerobic and BACTEC NR 6A bottles and 5,992 comparisons were made between BacT/Alert anaerobic and BACTEC NR 7A bottles. Each bottle was inoculated with 5 ml of blood. The overall recoveries of microorganisms from the two aerobic bottles were comparable; members of the family Enterobacteriaceae were recovered more often from BacT/Alert aerobic bottles alone (P less than 0.001). The overall recoveries of microorganisms from the anaerobic bottles were not significantly different. Growth of Staphylococcus aureus (P less than 0.001), coagulase-negative staphylococci (P less than 0.01), streptococci (P less than 0.001), Escherichia coli (P less than 0.01), other members of the family Enterobacteriaceae (P less than 0.02), and Pseudomonas aeruginosa (P less than 0.05) was detected earlier in BacT/Alert aerobic bottles. Growth of S. aureus (P less than 0.001), coagulase-negative staphylococci (P less than 0.05), enterococci (P less than 0.01), Streptococcus pneumoniae (P less than 0.02), viridans group streptococci (P less than 0.05), E. coli (P less than 0.001), Klebsiella pneumoniae (P less than 0.01), and other members of the family Enterobacteriaceae (P less than 0.001) was detected earlier in BacT/Alert anaerobic bottles. In a system-versus-system comparison, more gram-positive cocci were recovered from the BACTEC system alone (P < 0.05), and more members or the family Enterobacteriaceae were recovered from the BacT/Alert system alone (P < 0.001). As a system, the BacT/Alert system detected growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.01), streptococci (P < 0.001), E. coli (P < 0.001), other members of the familyEnterobacteriaceae (P < 0.001), and P. aeruginosa (P < 0.05) earlier than the BACTEC system did. Significantly fewer (40 versus 1,183) false-positive results occurred with the BacT/Alert system. We conclude that the BacT/Alert and BACTEC 660/730 nonradiometric systems are comparable for recovering clinically significant microorganisms form adult patients with bacteremia or fungemia, but that the BacT/Alert system detects microbial growth earlier than the BACTEC system does, with significantly fewer false-positive results.

Adult↗

PubMed1995

Comparison of microbial tests for the detection of heavy metal genotoxicity.

Heavy metal genotoxicity was evaluated by using different microbial tests. Four genotoxicity assays were employed: the Ames test, the E. coli WP2 test, the Mutatox test detecting mutagenicity, and the SOS assay with E. coli-detecting enzyme induction. All the metals tested (cadmium, chromium, copper, mercury, nickel, and zinc) were detected as genotoxic by the Mutatox and the SOS tests. The Ames test and the E. coli WP2 assay only detected chromium as genotoxic, causing a mutagenic effect. The sensitivity to metals of all the assays used was maintained when they were dissolved in sewage, although there was a slight increase in the sensitivity thresholds.

Environmental Exposure↗

PubMed2004

Development of rationally designed nucleic acid signatures for microbial pathogens.

The detection and identification of microbial pathogens are critical challenges in clinical medicine and public health surveillance. Advances in genome analysis technology are providing an unprecedented amount of information about bacterial and viral organisms, and hold great potential for pathogen detection and identification. In this paper, a rational approach to the development and application of nucleic acid signatures is described based on phylogenetically informative sequence features, especially single nucleotide polymorphisms. The computational tools that are available to enable the development of the next generation of microbial molecular signatures for clinical diagnostics and infectious disease surveillance are reviewed and the impact on public health and national security will be discussed.

Bacteria↗

PubMed1994

Transfusion-related transmissible diseases: detection by polymerase chain reaction-amplified genes of the microbial agents.

The detection of blood-borne microbes by PCR has broadly and rapidly progressed in the past 5 years as briefly described in this article. This progress has been largely because of the scientific developments made at Roche Molecular Systems by Sninsky et al through collaborations with academic and Government institutions. This unprecedented cooperation propels the continuing work at Roche Molecular Systems to bring the PCR technology into routine laboratory diagnosis. Whether the success of EIA in virtual elimination of hepatitis and retroviral infections can be matched by the cost-effectiveness of putative application of PCR in screening blood supply remains to be determined.

Bacterial Infections↗

PubMed1999

["Microbial air pollutants--methods for detection of endotoxins, mycotoxins and MVOC"].

In the context of microbial emissions from composting facilities the methods for the detection and identification of the groups of substances released, i.e. endotoxins, mycotoxins and Microbial Volatile Compounds (MVOC) are discussed. With the aid of an overview of the different methods employed for the investigation of the single groups of compounds the current state of the art in this field is presented. In conclusion the enormous research needs, especially with regard to the mycotoxins and MVOC, are pointed out.

Air Microbiology↗

PubMed1999

From field barley to malt: detection and specification of microbial activity for quality aspects.

Barley grain carries a numerous, variable, and complex microbial population that mainly consists of bacteria, yeasts, and filamentous fungi and that can partly be detected and quantified using plating methods and microscopic and molecular techniques. The extent and the activity of this microflora are determined by the altering state of the grain and the environmental conditions in the malt production chain. Three ecological systems can be distinguished: the growing cereal in the field, the dry barley grain under storage, and the germinating barley kernel during actual malting. Microorganisms interact with the malting process both by their presence and by their metabolic activity. In this respect, interference with the oxygen uptake by the barley grain and secretion of enzymes, hormones, toxins, and acids that may affect the plant physiological processes have been studied. As a result of the interaction, microorganisms can cause important losses and influence malt quality as measured by brewhouse performance and beer quality. Of particular concern is the occurrence of mycotoxins that may affect the safety of malt. The development of the microflora during malt production can to a certain extent be controlled by the selection of appropriate process conditions. Physical and chemical treatments to inactivate the microbial population on the barley grain are suggested. Recent developments, however, aim to control the microbial activity during malt production by promoting the growth of desirable microbial cultures, selected either as biocontrol agents inhibiting mycotoxin-producing molds or as starter cultures actively contributing to malt modification. Such techniques may offer natural opportunities to improve the quality and safety of malt.

Bacteria↗

PubMed1994

The value of amniotic fluid interleukin-6, white blood cell count, and gram stain in the diagnosis of microbial invasion of the amniotic cavity in patients at term.

PROBLEM: Subclinical microbial invasion of the amniotic cavity occurs in 18.8% of women with term labor and intact membranes and in 34% of patients with term PROM and is a risk factor for the development of puerperal infection related morbidity. Although amniotic fluid white blood cell count, interleukin-6 determination, and Gram stain examination have been used for the diagnosis of intrauterine infection in patients with preterm labor and preterm premature rupture of membranes, no information is available about the accuracy and specific cut-off values for these tests in patients at term. The purpose of this study was to compare the performance of the amniotic fluid Gram stain examination, white blood cell count, and interleukin-6 determination in the identification of microbial invasion of the amniotic cavity in patients at term with and without PROM. METHOD: Amniotic fluid was retrieved from 148 patients with term gestations (90 patients with spontaneous labor and intact membranes and 58 patients with PROM). Samples were cultured for bacteria and Mycoplasma species. Amniotic fluid Gram stain, white blood cell count, and interleukin-6 determinations (ELISA, sensitivity: 43 pg/ml) were performed in all samples. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture for microorganisms. Analysis was conducted using Mann-Whitney U test, Fisher's exact test, receiver operating characteristic curves and logistic regression. RESULTS: Patients with spontaneous labor and intact membranes: The prevalence of microbial invasion of amniotic cavity in this group was 15.6% (14/90). The most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determination (sensitivity for: interleukin-6 > or = 5.7 ng/ml = 86%, white blood cell count > or = 20 cells/mm3 = 64%, Gram stain = 28%). The most specific test was the Gram stain of the amniotic fluid (specificity for: Gram stain = 84%, interleukin-6 = 79% and white blood cell count = 63%). Multiple logistic regression demonstrated that amniotic fluid interleukin-6 concentration was the only covariate that retained statistical significance when intrauterine infection was used as outcome variable. Patients with PROM: The prevalence of a positive amniotic fluid culture in this group was 39.7% (23/58). Logistic regression demonstrated that only interleukin-6 retained a significant relationship with the results of amniotic culture when all variables were entered simultaneously into a model to predict amniotic fluid culture results. The most sensitive tests for the detection of intrauterine infection were interleukin-6 determination and white blood cell count (sensitivity for interleukin-6 > or = 3.4 ng/ml and white blood cell count > or = 20 cells/mm3 = 69.6% for both). The most specific test was Gram stain (97.1%). CONCLUSIONS: Amniotic fluid interleukin-6 determination is the best rapid test for the detection of microbial invasion of the amniotic cavity in patients at term with and without PROM. When this test is not available, amniotic fluid Gram stain and white blood cell count represent valid diagnostic tools to assess the microbial state of amniotic cavity.

Amniotic Fluid↗

PubMedDate not supplied

[Comparative study of microbial methods for the detection of genetic toxicity].

It is important to have a rapid and accurate method to detect the toxic action of drugs and chemical compounds used by man. A comparative study with two microbial systems was carried out: one using Salmonella typhimurium and the other using Bacillus subtilis. The 1-8-dihydroxyantraquinone was the tested drug and the N-methyl-N'-nitro-N-nitrosoguanidine and the 2-aminofluorene were used as control substances. These compounds were used as such or after previous transformation by a microsomal system. The results obtained showed that both systems: the S. typhimurium and the B. subtilis work in a similar way, but the latter allows a more direct action of drug on the genetic material. The effect of the solvents: ethanol and dimethylsulfoxide was analysed, because they affected the transformation and reversion processes that were carried out.

Anthraquinones↗

PubMed1996

Ampicillin and amoxicillin residue detection in milk, using microbial receptor assay (Charm II) and liquid chromatography methods, after extra-label administration of the drugs to lactating cows.

OBJECTIVE: A microbial receptor assay method (MRAM; Charm II test) for beta-lactam antibiotics and a liquid chromatography (LC) method with a detection limit of 2 to 5 ppb were evaluated for detection of ampicillin or amoxicillin residues in milk samples from individual cows. DESIGN: The MRAM was compared to the LC in 2 respects. Measured concentrations of drugs were compared, as well as the classification of samples relative to the FDA tolerance value of 10 ppb. ANIMALS: A total of 6 clinically normal lactating Holstein cows were used per drug. PROCEDURE: Ampicillin trihydrate or amoxicillin trihydrate was administered at an extra-label dosage of 22 mg/kg of body weight, IM, once to each of 6 cows/drug. Milk samples were collected at milkings prior to and for 156 hours after drug administration. Drug concentrations in milk samples from individual cows were determined by use of the MRAM and LC tests. Additionally, the classification of milk samples relative to the presence or absence of residues above the FDA tolerance value was determined. Pharmacokinetic analysis was performed on derived milk drug concentrations. RESULTS: Concentration of ampicillin in milk samples from all cows was < 10 ppb by the MRAM and LC methods by the fourth milking (48 hours) after treatment with ampicillin. Values were < 10 ppb by both methods for all cows treated with amoxicillin by the sixth milking (72 hours) after treatment. For individual milk samples, significant differences were found between test methods in the proportion of positive (failing) tests; the MRAM had a higher proportion of presumptive positives. CONCLUSIONS: Even at an extra-label dosage of 22 mg/kg, IM, milk residues > 10 ppb (the FDA tolerance value) were not detected beyond the label milk withholding times for ampicillin (48 hours) and amoxicillin (96 hours). When used for testing milk of individual cows by the control point procedure, the MRAM had a tendency to give presumptive positive test results for milk samples containing < 10 ppb ampicillin or amoxicillin as determined by LC.

Amoxicillin↗

PubMedDate not supplied

Biosensors for the analysis of food- and waterborne pathogens and their toxins.

Biosensors are devices which combine a biochemical recognition element with a physical transducer. There are various types of biosensors, including electrochemical, acoustical, and optical sensors. Biosensors are used for medical applications and for environmental testing. Although biosensors are not commonly used for food microbial analysis, they have great potential for the detection of microbial pathogens and their toxins in food. They enable fast or real-time detection, portability, and multipathogen detection for both field and laboratory analysis. Several applications have been developed for microbial analysis of food pathogens, including E. coli O157:H7, Staphylococcus aureus, Salmonella, and Listeria monocytogenes, as well as various microbial toxins such as staphylococcal enterotoxins and mycotoxins. Biosensors have several potential advantages over other methods of analysis, including sensitivity in the range of ng/mL for microbial toxins and <100 colony-forming units/mL for bacteria. Fast or real-time detection can provide almost immediate interactive information about the sample tested, enabling users to take corrective measures before consumption or further contamination can occur. Miniaturization of biosensors enables biosensor integration into various food production equipment and machinery. Potential uses of biosensors for food microbiology include online process microbial monitoring to provide real-time information in food production and analysis of microbial pathogens and their toxins in finished food. Biosensors can also be integrated into Hazard Analysis and Critical Control Point programs, enabling critical microbial analysis of the entire food manufacturing process. In this review, the main biosensor approaches, technologies, instrumentation, and applications for food microbial analysis are described.

Biosensing Techniques↗

PubMed2003

Taxonomic changes of bacteria associated with endodontic infections.

There have been major recent reorganizations among bacterial taxa as a result of phylogenetic taxonomic approaches. As a consequence, old species have been renamed and novel species have been proposed. The introduction of molecular technology for microbial identification has also allowed the detection of microbial taxa never previously found in endodontic infections. Therefore, the list of putative endodontic pathogens is frequently changing and expanding. The purpose of this review is twofold: to cover the taxonomic changes that the major putative endodontic pathogens have undergone in the recent years and to compile data from studies regarding the detection of known or novel bacterial species that had been only recently reported to occur in endodontic infections.

Bacteria, Anaerobic↗

PubMed2002

Microbiota of periapical lesions refractory to endodontic therapy.

The periapical microbiota of 36 teeth with refractory apical periodontitis was investigated. None of the teeth had responded to conventional endodontic or long-term (> 6 months), calcium-hydroxide treatment. Eight patients had received antibiotics systemically. After anaerobic culture, a total of 148 microbial strains were detected among 67 microbial species. One of the 36 lesions was culture-negative. Approximately half (51.0%) of the bacterial strains were anaerobic. Gram-positive species constituted 79.5% of the flora. Facultative organisms, such as Staphylococcus, Enterococcus, Enterobacter, Pseudomonas, Stenotrophomonas, Sphingomonas, Bacillus, or Candida species were recovered from 27 of the lesions (75%). Sulfur granules were found in 9 lesions (25%). In these granules Actinomyces israelii, A. viscosus, A. naeslundii, and A. meyeri were identified. Other bacterial species, both gram-positive and gram-negative, were detected in the granules as well. Two sulfur granules did not contain Actinomyces. Scanning electron microscopy demonstrated rod- and spirochete-like cells in the granules, and transmission electron microscopy revealed organisms with copious amounts of extracellular material. Outer membrane vesicles were also seen. Some of the granules were calcified. This study demonstrated a wide variety of microorganisms, particularly gram-positive ones, in the periapical lesions of teeth with refractory apical periodontitis.

Actinomyces↗

PubMed2000

Histamine and biogenic amine production by Morganella morganii isolated from temperature-abused albacore.

Histamine-producing bacteria were isolated from albacore stored at 0, 25, 30, and 37 degrees C. They were screened using Niven's differential medium, and their histamine production was confirmed by high-pressure liquid chromatography analysis. The optimum temperature for growth of histamine-producing bacteria was 25 degrees C. The bacterium producing the highest level of histamine was isolated from fish abused at 25 degrees C. It was identified as Morganella morganii by morphological, cultural, biochemical, and antimicrobial characteristics and by the Vitek microbial identification system. The M. morganii isolate was inoculated into tuna fish infusion broth medium, and the effect of temperature was determined for microbial growth and formation of histamine and other biogenic amines. The isolate produced the highest level of histamine, 5,253 ppm, at 25 degrees C in the stationary phase. At 15 degrees C, histamine production was reduced to 2,769 ppm. Neither microbial growth nor histamine formation was detected at 4 degrees C. To determine whether the isolate can also produce other biogenic amines that can potentiate histamine toxicity, production of cadaverine, putrescine, serotonin, tryptamine, tyramine, phenylethylamine, spermidine, and spermine by the isolate was also monitored. Cadaverine, putrescine, and phenylethylamine were detected with microbial growth in the tuna fish infusion broth medium. The optimum temperature for cadaverine, putrescine, and phenylethylamine formation was found to be 25 degrees C, as it was for histamine.

Animals↗

PubMed1994

Zymographic techniques for detection and characterization of microbial proteases.

We have presented a variety of zymographic techniques for identification and characterization of microbial proteases, using SDS-PAGE and PAGE in nondissociating gels. Techniques are described using copolymerized protein substrates, diffusable protein substrates, protein substrates incorporated into indicator gels, as well as synthetic esterase substrates. When a newly discovered protease is being characterized, it is advisable to try a variety of techniques, both to determine optimal conditions for enzyme detection and to characterize the protease. Zymography is a versatile two-stage technique involving protein separation by electrophoresis followed by detection of proteolytic activity. Each particular combination of protease separation and detection techniques had advantages and limitations. Protease separation by SDS-PAGE has as a limitation the fact that some proteases do not renature and hence cannot be detected following treatment with SDS. However, it has an advantage the fact that it allows estimation of the relative molecular weight of proteases. Protein separation using nondissociating PAGE is performed using much gentler protease inactivation conditions than those produced by treatment with SDS. Like SDS-PAGE, nondissociating PAGE permits detection of multiple forms of enzymes; however, a disadvantage is that it cannot be used to obtain molecular weight estimates of proteases. The main variable to control during development of zymograms is the length of time of incubations. Increasing incubation (development) time generally increases the sensitivity of protease detection; however, as the length of time of incubation increases so does the extent of diffusion of proteases and substrates. If incubations are prolonged, protease bands will diffuse, decreasing resolution. Additionally, zones of lysis produced by closely migrating proteolytically active species will merge, eliminating the possibility of detecting all proteolytic species in the sample. Zymographic techniques can be extremely useful in identification and characterization of microbial proteases. If a few properties of a protease are known, such as the pH range over which the enzyme is active, and whether it can renature after exposure to SDS, zymographic techniques can be specifically and readily adapted to optimize conditions for detection and assist in characterization of the enzyme.

Amino Acid Sequence↗

PubMed2003

The first fluorogenic assay for detecting a Baeyer-Villigerase activity in microbial cells.

The first fluorogenic assay allowing for detection of microbial enzymes able to perform Baeyer-Villiger oxidation is described. This is based on the use of 4-oxopentyl umbelliferyl ether 1 as a fluorogenic substrate. When Baeyer-Villigerases active against this test ketone are present in the selected whole cells, 1 is transformed into 3-hydroxypropyl umbelliferyl ether 3, which, in a subsequent step, releases the fluorescent product umbelliferone. Different microorganisms, known to be endowed with Baeyer-Villigerase activity, were assayed.

Bacteria↗

PubMed1993

The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and gram stain in patients with preterm labor and intact membranes.

OBJECTIVE: Our goal was to compare the value of amniotic fluid tests in the detection of microbial invasion of the amniotic cavity and in the relationship with the amniocentesis-to-delivery interval and neonatal complications in patients with preterm labor and intact membranes. STUDY DESIGN: Amniotic fluid was retrieved by transabdominal amniocentesis from 120 patients with preterm labor and intact membranes. Fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas. Amniotic fluid analysis included a Gram stain, white blood cell count, glucose and interleukin-6 determinations. Logistic regression and Cox's proportional hazards model were used for analysis. RESULTS: (1) The prevalence of positive amniotic fluid cultures was 9.2% (11/120); (2) patients with microbial invasion had a shorter amniocentesis-to-delivery interval and a higher neonatal complications rate than patients with a negative culture; (3) the most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determinations (cutoff 11.3 ng/ml) (sensitivity; for interleukin-6 100%, for glucose 81.8%, for white blood cell count 63.6%, and for Gram stain 63.6%; p < 0.05 for all comparisons); (4) the most specific test was the Gram stain of amniotic fluid (specificity: for Gram stain 99.1%, for white blood cell count 94.5%, for interleukin-6 82.6%, and for glucose 81.6%; p < 0.01 for all); (5) of all amniotic fluid tests, interleukin-6 determinations were the only ones that had significant relationship with the amniocentesis-to-delivery interval and neonatal complications. CONCLUSION: Interleukin-6 concentrations in amniotic fluid are better indicators of microbial invasion of the amniotic cavity, amniocentesis-to-delivery interval, and neonatal complications than the amniotic fluid Gram stain, glucose concentration, or white blood cell count.

Adult↗

PubMedDate not supplied

Microbial receptor assay for rapid detection and identification of seven families of antimicrobial drugs in milk: collaborative study.

A microbial competitive receptor assay for detecting residues of antibiotic families in milk was studied collaboratively by 13 laboratories. The drugs and levels (ppb) tested in this study include penicillin G, 4.8; cephapirin, 5.0; cloxacillin, 100; tetracycline, 2000; chlortetracycline, 2000; oxytetracycline, 2000; erythromycin, 200; lincomycin, 400; clindamycin, 400; sulfamethazine, 75; sulfamethoxazole, 50; sulfisoxazole, 50; streptomycin, 1000; novobiocin, 50; and chloramphenicol, 800. In this method, microbial cells added to a milk sample provide specific binding sites for which 14C or 3H labeled drug competes with drug residues in the sample. The 14C or 3H binding to the specific binding sites is measured in a scintillation counter and compared with a zero standard milk. If the sample is statistically different from the zero standard, it is positive. The assay takes about 15 min. The binding reaction occurs between the receptor site and the drug functional group, so all members of a drug family are detected. In this case, beta-lactams, tetracyclines, macrolides, aminoglycosides, novobiocin, chloramphenicol, and sulfonamides, including p-aminobenzoic acid (PABA) and its other analogs, are detectable. The incidence of false negative determinations among samples is about 1%; the incidence of false positives is about 3%. For negative cases, the relative standard deviations for repeatability ranged from 0 to 5% and for reproducibility from 0 to 6%. For positive cases, relative standard deviations ranged from 0 to 13% for repeatability and from 0 to 14% for reproducibility. The method has been adopted official first action.

4-Aminobenzoic Acid↗

PubMed2004

GC fractionation enhances microbial community diversity assessment and detection of minority populations of bacteria by denaturing gradient gel electrophoresis.

Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.

Animals↗