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PubMed1987

Rapid methods for the molecular diagnosis of infectious diseases: current trends and applications.

Methods for the rapid diagnosis of infectious diseases have become increasingly common in the last decade. The impetus for the development of such techniques has stemmed from the need to provide clinically relevant information without the length of time and complexity inherent to traditional cultivation methods. This is particularly important in high-risk populations for which more effective antibiotics and antiviral compounds are now available. Assay systems for the immunological detection of microbial agents occupy a central role in the molecular diagnosis of infectious diseases and are rapidly appearing on the market. Novel methods of organism detection by nucleic acid hybridization, long considered usable only in highly specialized laboratories, have the potential for use in routine microbiological laboratories and some systems are now commercially available. As more efficient and rapid diagnostics systems are being developed, the selection of the optimal method will depend on the environment in which the system is to be used. Rapid methods for the detection of infectious agents might markedly improve health care in a variety of clinical, laboratory and epidemiologic situations.

Agglutination Tests↗

PubMed2002

Use of polarography as a quality-control method for determining diacetyl in citrus and vegetable juices, yoghurt and butter.

The determination of diacetyl permits the detection of microbial growth in the processing of citrus fruit before the appearance of other organoleptic, chemical or microbiological changes. It also makes it possible to detect a break in the cold chain during distribution and sale. The study proposed a polarographic method for the determination of diacetyl that allowed routine analysis with the aim of detecting possible contamination in the citrus juice manufacturing chain (orange and orange-carrot). The analytical performance of the method in terms of a linearity from 0 to 960 microg ml(-1), a recovery of 97 to 98%, a precision of 3.2 to 4.8%, and a sensitivity of 0.2 ng ml(-1) for juices indicated the robustness of the method. The same method was also applied to butter and yoghurt with detection limits of and 0.4 ng g(-1).

Beverages↗

PubMed1976

Anaphylactoid reactions in connection with infusion of invert sugar solutions are due to macromolecular contaminants.

13 untoward mold anaphylactoid reactions were observed in patients infused with invert sugar solutions in Sweden during a 7-month period in 1973: an incidence of 1/31,000 infusions. Immunological and physicochemical analysis of invert sugar solutions and of the raw material, sucrose, revealed traces of native alpha-1,6-glucan with molecular weight of 10-100 millions as contaminant. This indicates, that the sucrose had been exposed to microbial contamination during its manufacture from sugar-beet or sugar-cane. Reversed single radial immunodiffusion was used for alpha-1,6-glucan detection and screening of all sucrose batches. Rejection of contaminated sucrose as raw material reduced the incidence of anaphylactoid invert sugar reactions to about 1/575,000 infusions. Examination of all sucrose raw material for traces of crude alpha-1,6-glucan is recommended as a test for detection of microbial contamination.

Anaphylaxis↗

PubMed2003

Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds.

A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P. putida F1 cym operon. Exposure of strain UT93 to 0.02-850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h. Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels. The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.

Air Microbiology↗

PubMedDate not supplied

Time course of radiometric detection of positive blood cultures in childhood.

We have determined the time course of radiometric detection of microbial growth in 2348 positive blood culture specimens obtained at Wyler Children's Hospital during a 5-year interval. Overall 72 and 88% of isolates were detected within 48 and 72 hours after sampling, respectively. For pathogenic organisms aerobic detection was generally more rapid and more inclusive than anaerobic detection. At 48 hours of incubation the detection of six potential pathogens (Salmonella sp., Haemophilus influenzae, Group D streptococci, Neisseria meningitidis, coagulase-negative staphylococci, Candida sp.) was significantly delayed compared with detection of other pathogenic organisms recovered from blood. At 72 hours of incubation the detection rates remained less than 95% for H. influenzae, Staphylococcus aureus, Klebsiella sp., coagulase-negative staphylococci, Group D streptococci and Candida sp. These data should assist clinical decisions regarding duration of antibiotic therapy for the presumptive diagnosis of bacteremia in children.

Anti-Bacterial Agents↗

PubMed2006

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents.

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

4-Butyrolactone↗

PubMed2002

Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass.

We have applied bioluminescent ATP detection methods to microbial enumeration in Antarctic Dry Valley mineral soils, and validated our ATP data by two independent methods. We have demonstrated that ATP measurement is a valid means of determining microbial biomass in such sites, and that the desiccated surface mineral soils of the Antarctic Dry Valleys contain cell numbers over four orders of magnitude higher than previously suggested.

Adenosine Triphosphate↗

PubMed1980

Effects of alkaline phosphatase activity on nucleotide measurements in aquatic microbial communities.

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.

Journal Article↗

PubMed2003

[Advances in the research of soil microorganisms and their mediated processes under heavy metal stress].

More and more evidence from both laboratory and field experiments suggested that soil microbes were more sensitive to heavy metal stress than soil animals or plants living in the same soil. This understanding has been gradually accepted as a common point in this field. The history and the latest advances in soil microorganisms and its mediated processes under heavy metal stresses in soils were reviewed in this paper. It is difficult to do any comparison among the results from laboratory, field toxicological experiment and environmental monitoring. So far, a relevantly 'accurate' control soil was rarely available in the environmental monitoring. Much of evidence has indicated that not only microbial population/biomass was changed under heavy metal stress, but also its structure was varied. Although many molecular methods, such as PLFA, BIOLOG and DNA, have been developed to detect this microbial structural change, they always need many expensive instruments and accessories, and only could be done in laboratory. Therefore, some rapid and easy methods are expected to develop to substitute the traditional plate count. The speciation in the solution phase, ion effect and rhizospheric effect of heavy metal on the toxicity to soil microorganisms and its mediated processes need extent studies and discussion. Finally, the microbial indicator system of assessing heavy metal toxicity to soil microorganisms and its mediated processes need to be established.

Bacteria↗

PubMed2000

The Toll receptor family and microbial recognition.

The survival of multicellular organisms is dependent on their ability to recognize invading microbial pathogens and to induce a variety of defense reactions. Recent evidence suggests that an evolutionarily ancient family of Toll-like receptors plays a crucial role in the detection of microbial infection and the induction of immune and inflammatory responses.

Animals↗

PubMed2006

The effectiveness of selected microbial and chemical indicators to detect sewer leakage impacts on urban groundwater quality.

Sewer and stormwater pipe leakage can lead to the degradation of urban groundwater quality. This groundwater may be subsequently used for public water supply and so the resulting water treatment and public health consequences can be serious. To understand the impact of sewer exfiltration on groundwater quality, suitable indicators need to be sampled and analysed for. This study examined potential sewer-derived inorganic and microbial parameters in the U.K. city of Doncaster. Sulphite reducing clostridia, faecal streptococci and boron were all detected in groundwater with reductions compared with sewer values ranging from 1 to 6 orders of magnitude for the former two, to 1 to 2 orders of magnitude decrease for boron. The correlation between these two different indicator types suggests that groundwater quality is being adversely affected by sewer leakage in the study area. The employment of several and varied indicators can better demonstrate the effect than use of single parameters.

Cities↗

PubMed2006

Metabolite production of yeasts on a strawberry-agar during storage at 7 degrees C in air and low oxygen atmosphere.

Changes of different quality factors of strawberries have been described until now from a physiological point of view. Possible effects on quality caused by yeast proliferation have not been described. To elucidate the metabolic activity of yeasts (i.e. Debaryomyces melissophilus, Rhodotorula glutinis, Cryptococcus laurentii), isolated from strawberries, they were inoculated on a simulation medium of strawberries (strawberry-agar). Their activity was measured by analysing and correlating microbiological counts, metabolite concentration in the headspace as well as in the medium, and sugar consumption. The isolated yeasts from strawberries could grow on the strawberry-agar, both under air conditions and modified atmosphere (MA) conditions. The maximum count for the yeasts reached 7.5-8.5 log cfu cm(-2) (air conditions) and 5.6-6.4 log cfu cm(-2) (MA conditions). Production or consumption of a number of compounds could be detected when microbial counts reached levels between 4.7 log cfu cm(-2) and 8.5 log cfu cm(-2) depending on species and atmospheric conditions. A range of volatile organic compounds, produced by the yeasts, was detected: acetone, ethyl acetate, ethanol, isopropyl acetate, ethyl butyrate, 1-propanol, 2-methyl-1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-hexanol and hexyl acetate. These compounds are able to influence the sensory properties of strawberries. A simultaneous decrease in sugar concentrations (sucrose, glucose, fructose) was observed. When ethanol reached high concentrations, ethyl acetate and ethyl butyrate were produced. This production can be attributed to a detoxification of ethanol by yeasts. The fermentative metabolism of yeasts during aerobic conditions could be explained by the Crabtree effect. As the detected volatile organic compounds produced by yeasts are also found in fresh strawberries, it can be concluded that these compounds are produced both by microbiological and physiological processes.

Colony Count, Microbial↗

PubMed1992

HBsAg in urine: a new approach for the detection of urinary antigens.

In order to define the optimal conditions for detection of microbial antigens in urine, urinary HBsAg excreted during hepatitis B was chosen as a model. Using commercial kits, which mainly involve anti-discontinuous epitopes, we found urinary HBsAg in only 50% of patients with HBsAg in their sera. In contrast, with an inhibition method involving a monoclonal antibody recognizing a continuous epitope, urinary HBsAg was found in 100% of these patients. Structural analysis of HBsAg showed that urinary HBsAg is denatured; it can escape detection by commercial kits well fitted for detection of native serum HBsAg. General implications for the revelation of urinary microbial antigens are discussed.

Antibodies, Monoclonal↗

PubMed2026

Probiotic supplementation increases fecal TLR4 agonists without improving disease activity in juvenile idiopathic arthritis: a randomized placebo-controlled trial.

Gut dysbiosis has been implicated in the pathogenesis of juvenile idiopathic arthritis (JIA), suggesting that microbiota-targeted interventions may influence immune signalling during early immune development. We conducted the PERMAJI multicentre randomized, double-blind, placebo-controlled trial to evaluate the effects of probiotic supplementation (VSL#3) on host-microbiota immune interactions and disease activity in children with oligoarticular or RF-negative polyarticular JIA. Participants were randomly assigned (1:1) to receive VSL#3 or placebo for 3 months in addition to standard therapy. Stool and serum samples collected at baseline and month 3 were used to assess gut microbiota composition, fecal innate immune agonists, intestinal permeability, and systemic cytokines. The primary clinical endpoint was the proportion achieving an ACR Pedi 30 response at 3 months. Forty-four children were enrolled between September 2017 and July 2022. Clinical responses did not differ between groups (ACR Pedi 30: 47% with VSL#3 vs 63% with placebo; p = 0.33), and conservative worst-case assumptions for missing data suggested lower response rates with VSL#3 (36% vs 68%; p = 0.03). Probiotic supplementation significantly increased fecal Toll-like receptor 4 (TLR4) agonist activity, whereas gut microbiota diversity, intestinal permeability, and systemic cytokine levels remained unchanged. These findings indicate that probiotic supplementation can modify microbial innate immune signalling without detectable changes in microbial community diversity and may increase exposure to pro-inflammatory microbial stimuli in early-life autoimmune disease. The results highlight the complexity of host-microbiota immune interactions and underscore the need for careful evaluation of microbiome-targeted therapies in paediatric autoimmune disorders.

Humans↗

PubMed2000

Seasonal microbial community dynamics in a flowerpot-using personal composting system for disposal of household biowaste.

Microbial community dynamics in a flowerpot-using solid biowaste composting (FUSBIC) process were monitored seasonally by quinone profiling and conventional microbiological methods. The FUSBIC system, which consisted of three flowerpots (14 L or 20 L capacity) with 5-6 kg each of a soil-compost mixture (SCM) as the primary reactors, was loaded daily with household biowaste from November 1998 to October 1999. The monthly average waste reduction rate was 88.2% for the 14-L system and 92.5% for the 20-L system on a wet weight basis. The direct total microbial count detected in the 14-L primary reactors ranged from 4.5 to 9.6x10(11) cells.g(-1) of dry wt of SCM, and the viable count of aerobic heterotrophic bacteria recovered on agar plates at 28 degrees C varied from 1.9 to 5.7x10(11) CFU.g(-1) of dry wt. The quinone content of SCM samples from the 14-L and 20-L systems ranged from 160 to 353 nmol.g(-1) of dry SCM. Ubiquinones, unsaturated menaquinones, and partially saturated menaquinones constituted 15.0-36.4, 14.8-22.0, and 41.8-61.6 mol% of the total content, respectively. The major quinone types detected were usually MK-8(H(2)), MK-9(H(2)), and Q-10. Variations in quinone profiles were evaluated numerically by using two parameters, the dissimilarity index (D) and microbial divergence index (MD(q)). The upper limit of seasonal changes in the microbial community structure was about 30% as expressed by D values. The MD(q) values calculated ranged from 18 to 22. A significant positive correlation was found between seasonal temperature and bacterial populations containing partially saturated menaquinones. These results indicated that the FUSBIC system contained highly diverse microbial populations that fluctuated to some extent depending on seasonal temperature. Members of the Actinobacteria were suggested to be the major constituents of the total population present.

Journal Article↗

PubMed1999

PCR and blood culture for detection of Escherichia coli bacteremia in rats.

Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 10(6) CFU of Escherichia coli. Blood was then taken at several time points for detection of E. coli by BC and by PCR with E. coli-specific primers derived from the uidA gene, encoding beta-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after E. coli challenge. Without this chemotherapy, the E. coli detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (P, >0.05). Chemotherapy decreased the E. coli detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (P, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia.

Animals↗

PubMed2004

Nods and 'intracellular' innate immunity.

Innate immunity relies on the detection of microbial invaders by two distinct systems. One system comprises a family of membrane-bound receptors, termed the Toll-like receptors, while the other family, termed the nucleotide-binding site/leucine-rich repeat (NBS/LRR) proteins, consists of molecules that are found in the cytoplasmic compartment. These two detection systems recognize conserved molecular components of microbes including such structural motifs as lipopolysaccharide from the Gram-negative bacterial cell wall and peptidoglycan (PGN) found in the cell wall of both Gram-negative and Gram-positive bacteria. This review focuses on two members of the NBS/LRR family of proteins, Nod1 and Nod2. Recently, the microbial motifs sensed by these two molecules have been characterized. Both Nod1 and Nod2 recognize PGN, however, each requires distinct molecular motifs to attain sensing. Nod1 recognizes a naturally occurring muropeptide of PGN that presents a unique amino acid at its terminus called diaminopilemic acid (DAP). This amino acid is found mainly in the PGN of Gram-negative bacteria designating Nodl as a sensor of Gram-negative bacteria. In contrast, Nod2 can detect the minimal bioactive fragment of PGN, called muramyl dipeptide. Thus Nod2 is a general sensor of bacterial PGN. Since mutations in the gene encoding Nod2 were recently shown to be associated with the chronic inflammatory disease, Crohn's disease, these results are discussed in the context of how disrupting the interplay between host detection and bacterial aggression may lead to inflammatory diseases.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Development of a Gram-negative selective agar (GNSA) for the detection of Gram-negative microflora in sputa in patients with cystic fibrosis.

AIMS: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF). METHODS AND RESULTS: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30 g), bacteriological agar no.1 (10 g), yeast extract (5 g), crystal violet (2 mg), nisin (48 mg), novobiocin (5 mg), cycloheximide (100 mg), amphotericin (2 mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF. GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined. Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1.50 x 102 CFU ml-1 sputum Pseudomonas aeruginosa, 2.38 x 102 CFU ml-1 sputum Burkholderia cepacia genomovar IIIb and 6.70 x 103 CFU ml-1 sputum Stenotrophomonas maltophilia. A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection. CONCLUSIONS: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged. SIGNIFICANCE AND IMPACT OF THE STUDY: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.

Adult↗