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PubMed1962

Fibrinogen media for studies on staphylococci.

Deneke, Anneliese (University of Wisconsin, Madison) and Hans Blobel. Fibrinogen media for studies on staphylococci. J. Bacteriol. 83:533-537. 1962-Fibrinogen media were prepared simply by spreading rabbit plasma or a bovine fibrinogen solution over the surface of certain solidified agar media, thus eliminating the necessity of incorporation of fibrinogen into liquefied agar media at a critical temperature. With agar media composed of heart infusion broth, blood agar base, or Trypticase soy broth, distinct coagulase reactions were observed which corresponded closely to those obtained with the tube test.A bovine fibrinogen solution, applied to the surface of some of the commonly used selective media for staphylococci, provided an additional and more specific test system for coagulase production of individual colonies, and consequently aided in the presumptive identification of pathogenic staphylococci.

Animals↗
PubMed2006

Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

Ascomycota↗
PubMed2006

Development of a serotype-specific DNA microarray for identification of some Shigella and pathogenic Escherichia coli strains.

Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotype-specific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 10(4) CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.

Bacterial Typing Techniques↗
PubMed1986

Increased detection of prolylaminopeptidase in Neisseria meningitidis by Identicult-Neisseria.

Identicult-Neisseria (Scott Laboratories, Inc., Fiskeville, R.I.), a rapid enzymatic method with chromogenic substrates, was tested in our laboratories for the identification of Neisseria gonorrhoea, Neisseria meningitidis, and Neisseria lactamica. The test correlated very highly in its identification of pathogenic Neisseria spp. with modified New York City fermentation medium. Identicult-Neisseria appeared to be more sensitive in its detection of prolylaminopeptidase activity in N. meningitidis than most of the currently available systems.

Aminopeptidases↗
PubMed1986

Determination of molecular species composition of C80 or longer-chain alpha-mycolic acids in Mycobacterium spp. by gas chromatography-mass spectrometry and mass chromatography.

The molecular species composition of alpha-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular species of alpha-mycolic acids were well separated as trimethylsilyl ether derivatives of the methyl esters, according to their total carbon numbers. The total carbon and double-bond numbers of mycolic acids at each peak on gas chromatograms were determined from the [M]+, [M - 15]+, and [M - 90]+ ions on the mass spectrum, and straight and branched chain structures were identified by the mass fragment ions [A]+, due to C2--C3 cleavage [R-CH-O-Si(CH3)3]+, and [B]+, due to C3--C4 cleavage [(CH3)3-Si-O-CH-CH(R')-COOCH3]+. The concentration of odd- and even-carbon-numbered mycolic acids, which often overlap each other on gas chromatograms, and the composition of three homologous mycolic acids with different alpha units (C22:0, C24:0, and C26:0) were clearly determined by mass chromatography monitoring [M - 15]+ ions and [B - 29]+ ions, respectively. The molecular species composition of alpha-mycolic acids and their average carbon numbers (av. cn.) as a simple expression of the composition were calculated from the mass chromatograms. Each mycobacterial species examined was demonstrated to possess a characteristic profile of alpha-mycolic acid composition, and based on this the species were classified approximately into eight groups: C68 to C76 (av. cn. 72), dienoic, possessing a C20 alkyl branch at the 2 position (C22 alpha-unit) for Mycobacterium diernhoferi and Mycobacterium sp. strain 3707, a chromogenic rapid grower; C72 to C78 (av. cn. 75), dienoic with both C22 and C24 alpha units, containing a small or a large amount of odd-carbon-numbered molecules, for M. vaccae, M. rhodesiae, and M. phlei (chromogenic rapid growers); C72 to C80 (av. cn. 75 to 77), dienoic with C24 alpha-unit, containing a moderate or a large amount of odd-carbon-numbered molecules, for M. smegmatis, M. chitae, M. chelonae (M. chelonei), and M. fortuitum (nonchromogenic rapid growers); C78 to C82 (av. cn. 80), even-carbon-numbered dienoic with C24 alpha unit for M. agri and M. thermoresistible (rapid growers); C75 to C81 (av. cn. 77 to 79), odd-carbon-numbered dienoic with C24 alpha unit for M. nonchromogenicum complex (M. nonchromogenicum, M. terrae, and "M. novum") (slow growers); (vi) C76 to C84 (av. cn. 79 to 81), even-carbon-numbered dienoic with C24 alpha unit for MAIS complex including M. scrofulaceum, M. avium, and M. intracellulare (slow growers); (vii) C72 to C80 (av. cn. 77 to 79), even-carbon-numbered dienoic with C24 alpha unit for M. szulgai, M. gordonae, and M. kansasii (chromogenic slow growers); and (viii) C76 to C86 (av. cn. 79 to 81), even-carbon-numbered dienoic with C26 alpha unit M. bovis Ravenol and BCG and M. tuberculosis H37Rv. This study demonstrated that gas chromatography-mass spectrometric analysis of the molecular species composition of alpha-mycolic acid can give rapid, important, and very precise information for the identification of pathogenic and nonpathogenic mycobacterial species.

Chromatography, Gas↗
PubMed1989

T-mod pathway, a reduced sequence for identification of gram-negative urinary tract pathogens.

In this paper, we describe a reduced sequence of identification that includes T-mod medium, a selective and differential isolation medium which allows accurate presumptive identification of the most common gram-negative bacteria encountered in urine samples. The present study, performed on bacteria isolated from 1,762 independent urine samples, has shown that a few selected tests (lysine and ornithine decarboxylase, urease and trehalose fermentation tests) improve the identification accuracy of T-mod, making it possible both to identify the less frequent species and to prevent some misidentifications of Klebsiella pneumoniae and Proteus mirabilis. The proposed work flow agreed with conventional identification protocols to a 99.3% extent and allowed identification of 87.4% of the isolates directly from the primary plate, 11.4% after 1 to 3 additional tests, and 1.2% after an identification gallery.

Bacteriuria↗
PubMed1992

Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.

Base Sequence↗
PubMed2001

Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB).

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif(r) region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.

DNA Restriction Enzymes↗
PubMed2004

Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit.

Efficient, rapid, and reproducible procedures for isolating high-quality DNA before PCR gene amplification are essential for the diagnostic and molecular identification of pathogenic bacteria. This study evaluated the Qiagen QIAamp DNA Mini Kit and the Schleicher and Schuell IsoCode Stix DNA isolation device for isolating nucleic acid. Buffer, serum, and whole-blood samples were spiked with Bacillus anthracis Sterne vegetative cells and Yersinia pestis, while water was spiked with B. anthracis Sterne spores. Although minimal variations in limit of detection occurred among matrices, both the IsoCode Stix extraction method and the Qiagen procedure have comparable detection limits.

Bacillus anthracis↗
PubMed2026

Idiopathic neonatal arterial ischaemic stroke: a trio-based whole-exome sequencing study.

OBJECTIVE: To assess the contribution of rare coding genetic variants to idiopathic neonatal arterial ischaemic stroke (NAIS). DESIGN: Observational genetic study using trio-based whole-exome sequencing (WES). SETTING: Multicentre study. PATIENTS: 23 newborns diagnosed with idiopathic NAIS and their biological parents. INTERVENTIONS: WES-trio with a customised workflow for filtering and interpreting variants in de novo autosomal dominant and recessive inheritance models. MAIN OUTCOME MEASURES: Identification of pathogenic (P) or likely pathogenic (LP) variants potentially associated with NAIS. RESULTS: We identified 28 unique rare de novo variants in 28 genes across 23 newborns with NAIS. Under the autosomal recessive model, no candidate genes were identified. No common P/LP variant across the 23 newborns was detected. In-silico predictors and comprehensive knowledge-driven analysis highlighted PIK3CD (p.Gln431Arg) as a candidate gene in one patient with perforant stroke. However, no more cases were identified with PIK3CD variants, and functional studies are warranted to assess its pathogenicity impact. CONCLUSIONS: Trio-based WES did not identify a monogenic cause for idiopathic NAIS. Coding variants therefore appear unlikely to explain the underlying genetic base of the disease. Furthermore, PIK3CD (p.Gln431Arg) may contribute to perforant stroke, although it requires further association evidence. As the potential role of non-coding or structural variants in NAIS remains possible, genome-wide long-read sequencing approaches may provide further insights into the genetic architecture of this condition.

Humans↗
PubMed2001

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

BACKGROUND: Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood. AIM: To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes. METHODS: Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

Apoptosis↗
PubMed2000

Western blotting is useful in the salivary diagnosis of Helicobacter pylori infection.

BACKGROUND: The salivary diagnosis of Helicobacter pylori infection offers attractive possibilities for the epidemiological study of infection in children. Salivary enzyme linked immunosorbent assay (ELISA) is less reliable then serum ELISA, owing to variable transudation of immunoglobulin. In addition, children are more difficult to study because of lower specific serum antibody concentrations to H pylori. The performance of salivary western blotting in comparison with serum western blotting and serum ELISA was investigated in school children. SUBJECTS AND METHODS: Paired serum and saliva specimens were obtained from 669 [corrected] school children aged 9-11 in 10 British towns. All saliva and serum specimens were first analysed by ELISA; subsequently, western blotting of both specimens was performed on 31 and 34 specimens, respectively, to establish the criteria for positivity for western blotting. The remaining 121 specimens were then tested blindly and saliva was compared with the serum. RESULTS: The sensitivity and specificity of salivary ELISA in the 669 [corrected] specimens was 32 of 50 (64%) and 530 of 619 (86%) [corrected], respectively, when compared with serum ELISA. The western blotting validation was performed on 28 subjects with positive serum and positive salivary ELISA, 28 saliva positives with negative serum, 16 saliva negatives with positive serum, and 50 doubly negative subjects. Compared with serum western blots, the sensitivity and specificity of salivary western blots was 38 of 47 (81%) and 68 of 75 (91%), respectively. Using serum ELISA as the gold standard, the sensitivity and specificity were 32 of 44 (73%) and 72 of 78 (92%), respectively, the specificity being significantly higher than salivary ELISA (p < 0.001). CONCLUSION: Salivary western blotting for IgG is useful in the diagnosis of H pylori infection and is superior to ELISA. It also permits the identification of pathogenic strains.

Blotting, Western↗
PubMed2026

LDLR Variant Classification Through Activity-Normalized Prime Editing Screening.

BACKGROUND: Inherited variants in the LDL (low-density lipoprotein) receptor (LDLR) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. METHODS: We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. RESULTS: We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. CONCLUSIONS: Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.

CRISPR screening↗
PubMed1990

Receptors on airway gland cells.

Airway submucosal glands are by volume the most important source of macromolecules in airway secretions. These secretions, containing gel-forming mucins, antibacterial proteins, and antiproteases, comprise the major defensive barrier protecting the host against airborne pathogens. The identification of the mechanisms regulating secretion from the submucosal glands is key to understanding the genesis of this barrier and how it is altered by disease processes. Using a variety of methods, we and others have identified on the gland cells of several species receptors specific for ACh, norepinephrine, substance P, VIP, PGE1, PGE2, PGA1, PGD2, histamine and bradykinin. These receptors all participate in modulating the secretory activity of the airway submucosal glands. Studies of homogeneous cultures of bovine airway serous cells have yielded detailed information regarding the beta-adrenergic receptor on these cells. Using radioligand binding techniques, we found evidence for the presence of a single high affinity beta receptor of beta-2 subtype. Occupancy of this receptor by isoproterenol causes an elevation in the concentration of intracellular cAMP, which in turn stimulates the phosphorylation of a subset of cytoplasmic and membrane proteins. Based on the kinetics and pharmacology of these effects, it is likely that cAMP functions as a second messenger in the serous cell secretory pathway, probably acting through protein kinases. Current efforts are directed at identification of those phosphoproteins whose phosphorylation and dephosphorylation times are consistent with their possible roles in secretion.

Animals↗
PubMed1983

Rapid diagnosis of community-acquired bacterial pneumonia.

Rapid identification of pathogens in patients with bacterial pneumonia is important for optimal antimicrobial therapy. Coagglutination was compared with counterimmunoelectrophoresis (CIE) for sensitivity and specificity in the detection of Streptococcus pneumoniae, Hemophilus influenzae, Klebsiella pneumoniae, and Pseudomonas aeruginosa antigens in sputum specimens of 101 patients with community-acquired pneumonia. Coagglutination detected the appropriate bacterial antigen in 16 of 17 (94%) definite etiology patients and CIE detected 11 (64%). In 17 probable etiology patients, bacterial antigens were detected by coagglutination in 15 (88%) and by CIE in 7 (41%). Only 1 pathogen was falsely identified in the 19 culture-negative control patients, indicating a high degree of specificity for both tests. Coagglutination was more sensitive than CIE (p less than 0.05) or sputum stained with Gram's stain (p less than 0.05) in correctly identifying the putative pathogen in sputum. Our results strongly suggest that coagglutination is a useful diagnostic technique for establishing a rapid etiologic diagnosis in community-acquired pneumonia.

Agglutination Tests↗
PubMed1984

Free fatty acid and triglyceride content of Mycobacterium avium cultured under different growth conditions.

Previous investigations demonstrated that Mycobacterium avium has a requirement for fatty acid, which can be fulfilled by palmitic (C16:0) or oleic (C18:1) acids, and that it incorporates the fatty acid into triglycerides that are later utilized. Mycobacterium avium was grown in continuous culture or batch-cultured in medium that contained palmitic acid as the fatty acid source, but lacked albumin. Cells were extracted and free fatty acids and triglycerides were obtained by preparative thin-layer chromatography. The triglycerides were further purified by column chromatography. The free fatty acids were methylated and analyzed by gas chromatography. Oleic acid represented 40 to 64% of the total free fatty acids, except for cells batch-cultured and limited for nitrogen. The latter cells contained about 27% oleic acid, and 24% of the free fatty acids were of sizes greater than C24:0. The amount of palmitic acid varied considerably, but it and oleic acid together usually accounted for 70% of the total free fatty acids. The overall fatty acid content of the triglycerides was similar to that of the free fatty acids. However, two size classes of triglycerides were found that approximated the sizes of tristearin (C54) and tricaprylin (C24), molecular weights of 892 and 471 daltons, respectively. It is concluded that these types of studies may eventually lead to more accurate identification of pathogenic mycobacteria by means of gas chromatographic analyses.

Chromatography, Gas↗