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J R Johnson

Publications and source records attributed to J R Johnson.

At least 73 records · Page 4Linked to original sources
PubMed2000

Development of polymerase chain reaction-based assays for bacterial gene detection.

Polymerase chain reaction-based assays provide rapid, simple, and sensitive detection of bacterial genes, but are not without their drawbacks. This review summarizes the principal advantages and disadvantages of PCR-based bacterial gene detection, provides guidelines for the development and validation of new PCR assays, and describes potential pitfalls that may be encountered and how these can be avoided.

DNA Primers↗
PubMed2000

Possible role of CREB in the stimulation of oligodendrocyte precursor cell proliferation by neurotrophin-3.

We have previously shown that the transcription factor CREB (cyclic AMP-response element binding protein) could be a mediator of neuronal signals that, coupled to different signal transduction pathways, may play different regulatory roles at specific stages of oligodendrocyte (OLG) development. We have found before that in committed OLGs, CREB activation by phosphorylation can be triggered by beta-adrenergic stimulation and appears to play a role in the induction of OLG differentiation by cyclic AMP. In contrast, in OLG precursor cells, CREB phosphorylation is stimulated by neuroligands that increase calcium levels by a process that involves a mitogen-activated protein kinase (MAPK)/protein kinase C (PKC) pathway. This observation suggested that at this early developmental stage, CREB could play a role in regulating cell proliferation. In support of this hypothesis, we have now found that a rapid and dramatic stimulation of CREB phosphorylation is one of the earliest events that precedes the increase in cell proliferation that is observed when OLG precursors are treated with neurotrophin-3 (NT-3). Experiments in which CREB phosphorylation was investigated in the presence of different kinase inhibitors indicated that the activation of this transcription factor in the presence of NT-3 is mediated by the concerted action of MAPK- and PKC-dependent signal transduction pathways. Moreover, our present results also showed that down-regulation of CREB expression in the OLG precursors abolished the increase in DNA synthesis that is observed when the cultures are treated with NT-3. Thus, these results support the idea that in immature OLG precursors, CREB plays an important role in transducing signals which, like NT-3, may regulate cell proliferation.

Animals↗
PubMed2000

The final nail.

Explore the source record for details and available documents.

Antifungal Agents↗
PubMed2000

Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise.

Among 75 urosepsis isolates of Escherichia coli, 29 virulence factor (VF) genes were detected by use of a novel polymerase chain reaction (PCR) assay. Compared with probe hybridization, the PCR assay's specificity was 100% and sensitivity 97.1%. fyuA (yersiniabactin: overall prevalence, 93%), traT (serum resistance, 68%), and a pathogenicity-associated island marker (71%) occurred in most strains from both compromised and noncompromised hosts. Present in <20% of strains each were sfaS, focG (F1C fimbriae), afa/dra, bmaE (M fimbriae), gafD (G fimbriae), cnf1, cdtB (cytolethal distending toxin), cvaC (colicin V), and ibeA (invasion of brain endothelium). Different VFs were variously confined to virulence-associated phylogenetic group B2 (as defined by multilocus enzyme electrophoresis); concentrated in group B2, but with spread beyond; or concentrated outside of group B2. These findings provide novel insights into the VFs of extraintestinal pathogenic E. coli and demonstrate the new PCR assay's utility for molecular epidemiological studies.

Bacteremia↗
PubMed2000

Improved repetitive-element PCR fingerprinting of Salmonella enterica with the use of extremely elevated annealing temperatures.

Modified thermal cycling conditions were explored in an effort to improve the reproducibility and resolving power of repetitive-element PCR (rep-PCR) fingerprinting. Assay performance was rigorously evaluated under standard and modified cycling conditions, using as a test set 12 strains putatively representing 12 serovars of Salmonella enterica. For all three fingerprint types (ERIC2, BOXA1R, and composite fingerprints), the use of extremely elevated annealing temperatures plus an initial "touchdown" cycling routine yielded significant improvements in day-to-day reproducibility and discriminating power despite the somewhat sparser appearance of the fingerprints. Modified cycling conditions markedly reduced the variability of fingerprints between cyclers, allowing fingerprints from different cyclers to be analyzed together without the degradation of assay performance that occurred with between-cycler analyses under standard cycling conditions. With modified cycling, composite fingerprints exhibited the lowest reproducibility but the highest net discriminating power of the three fingerprint types. rep-PCR fingerprints led to the discovery of a serotyping error involving one of the 12 test strains. These data demonstrate that modified cycling regimens that incorporate elevated annealing temperatures (with or without an initial touchdown routine) may markedly improve the performance of rep-PCR fingerprinting as a bacterial typing tool.

DNA Fingerprinting↗
PubMed2000

Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli.

Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for Escherichia coli, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility. Using the ERIC2 and BOXA1R primers and 15 E. coli strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis [MLEE], including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships. Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution. The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions. Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2. These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic E. coli.

DNA Fingerprinting↗
PubMed2000

Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex PCR-based assay.

Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic Escherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of papA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the papA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that papA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of papA alleles between lineages. Sequencing of papA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.

Alleles↗
PubMed2000

Molecular epidemiological and phylogenetic associations of two novel putative virulence genes, iha and iroN(E. coli), among Escherichia coli isolates from patients with urosepsis.

Two novel putative Escherichia coli virulence genes, iha and iroN from E. coli (iroN(E. coli)), were detected in 55 and 39%, respectively, of 67 E. coli isolates from patients with urosepsis. iha and iroN(E. coli) exhibited divergent associations with other putative virulence genes, phylogenetic markers, host characteristics, and antimicrobial resistance.

Adult↗
PubMed2000

Evidence of commonality between canine and human extraintestinal pathogenic Escherichia coli strains that express papG allele III.

Although dogs have been proposed as carriers of extraintestinal pathogenic Escherichia coli (ExPEC) with infectious potential for humans, presumed host species-specific differences between canine and human ExPEC strains have cast doubt on this hypothesis. The recent discovery that allele III of papG (the P fimbrial adhesin gene) predominates among human cystitis isolates and confers an adherence phenotype resembling that of canine ExPEC prompted the present reevaluation of the canine-human ExPEC connection. Sixteen paired pap-positive urine and rectal E. coli isolates from dogs with urinary tract infection were studied. papG (adhesin) and papA (pilin) allele type, agglutination phenotypes, virulence factor genotypes, and randomly amplified polymorphic DNA and pulsed-field gel electrophoresis fingerprints were analyzed and compared with those of human ExPEC controls. The 16 canine strains contained predominantly papG allele III. Agglutination phenotypes segregated strictly according to papG allele status and were homogeneous among strains with the same papG allele profile irrespective of their human versus canine origin. Canine and human PapG variant III peptide sequences were highly homologous, without host species-specific differences. The most prevalent canine papA allele was F48, a novel variant recently identified among human urosepsis isolates. In addition to pap, human ExPEC-associated virulence genes detected among the canine strains included sfa/focDE, sfaS, fyuA, hlyA, cnf1, cdtB, kpsMT-II and -III, rfc, traT, ompT, and a marker for a pathogenicity-associated island from archetypal human ExPEC strain CFT073. Molecular fingerprinting confirmed the fecal origin of all but one canine urine isolate and showed one pair of O6 canine urine and fecal isolates to be extremely similar to an O6 human urosepsis isolate with which they shared all other genotypic and phenotypic characteristics analyzed. These data demonstrate that canine ExPEC strains are similar to, and in some instances essentially indistinguishable from, human ExPEC strains, which implicates dogs and their feces as potential reservoirs of E. coli with infectious potential for humans.

Adhesins, Escherichia coli↗
PubMed2000

Escherichia coli serotype O15:K52:H1 as a uropathogenic clone.

To clarify the clinical and bacteriological correlates of urinary-tract infection (UTI) due to Escherichia coli O15:K52:H1, during a 1-year surveillance period we prospectively screened all 1, 871 significant E. coli urine isolates at the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, for this serotype and assessed the epidemiological features of community-acquired UTI due to E. coli O15:K52:H1 versus other E. coli serotypes. We also compared the 25 O15:K52:H1 UTI isolates from the present study with 22 O15:K52:H1 isolates from other, diverse geographic locales and with 23 standard control strains (8 strains from the ECOR reference collection and 15 strains of nonpathogenic O:K:H serotypes) with respect to multiple phenotypic and genotypic traits. Although E. coli O15:K52:H1 caused only 1.4% of community-acquired E. coli UTIs during the surveillance period, these UTIs were more likely to present as pyelonephritis and to occur in younger hosts, with similar risk factors, than were UTIs due to other E. coli serotypes. Irrespective of geographic origin, E. coli O15:K52:H1 strains exhibited a comparatively restricted repertoire of distinctive virulence factor profiles (typically, they were positive for papG allele II, papA allele F16, and aer and negative for sfa, afa, hly, and cnf1), biotypes, ribotypes, and amplotypes, consistent with a common clonal origin. In contrast, their antimicrobial resistance profiles were more extensive and more diverse than those of control strains. These findings indicate that E. coli O15:K52:H1 constitutes a broadly distributed and clinically significant uropathogenic clone with fluid antimicrobial resistance capabilities.

Bacteremia↗