9 research outputs found
Meiotic Studies on Combinations of Chromosomes With Different Sized Centromeres in Maize
Multiple centromere misdivision derivatives of a translocation between the supernumerary B chromosome and the short arm of chromosome 9 (TB-9Sb) permit investigation of how centromeres of different sizes behave in meiosis in opposition or in competition with each other. In the first analysis, heterozygotes were produced between the normal TB-9Sb and derivatives of it that resulted from centromere misdivision that reduced the amounts of centromeric DNA. These heterozygotes could test whether these drastic differences would result in meiotic drive of the larger chromosome in female meiosis. Cytological determinations of the segregation of large and small centromeres among thousands of progeny of four combinations were made. The recovery of the larger centromere was at a few percent higher frequency in two of four combinations. However, examination of phosphorylated histone H2A-Thr133, a characteristic of active centromeres, showed a lack of correlation with the size of the centromeric DNA, suggesting an expansion of the basal protein features of the kinetochore in two of the three cases despite the reduction in the size of the underlying DNA. In the second analysis, plants containing different sizes of the B chromosome centromere were crossed to plants with TB-9Sb with a foldback duplication of 9S (TB-9Sb-Dp9). In the progeny, plants containing large and small versions of the B chromosome centromere were selected by FISH. A meiotic “tug of war” occurred in hybrid combinations by recombination between the normal 9S and the foldback duplication in those cases in which pairing occurred. Such pairing and recombination produce anaphase I bridges but in some cases the large and small centromeres progressed to the same pole. In one combination, new dicentric chromosomes were found in the progeny. Collectively, the results indicate that the size of the underlying DNA of a centromere does not dramatically affect its segregation properties or its ability to progress to the poles in meiosis potentially because the biochemical features of centromeres adjust to the cellular conditions
Multiplex Identification of Gram-Positive Bacteria and Resistance Determinants Directly from Positive Blood Culture Broths: Evaluation of an Automated Microarray-Based Nucleic Acid Test
<div><p>Background</p><p>A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria.</p><p>Methods and Findings</p><p>1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., <i>Listeria</i> spp., <i>S. lugdunensis</i>, <i>vanB</i>-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%–100% and 95.4%–100%, respectively. Identification of the <i>mecA</i> gene in 599 cultures containing <i>S. aureus</i> or <i>S. epidermidis</i> was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the <i>vanA</i> gene in 81 cultures containing <i>Enterococcus faecium</i> or <i>E. faecalis</i> was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant <i>S. aureus</i> and vancomycin resistant <i>Enterococcus</i> spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign <i>mecA</i> to a specific organism in cultures containing more than one <i>Staphylococcus</i> isolate and does not identify common blood culture contaminants such as <i>Micrococcus</i>, <i>Corynebacterium</i>, and <i>Bacillus</i>.</p><p>Conclusions</p><p>The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures.</p><p><i>Please see later in the article for the Editors' Summary</i></p></div
Detection of <i>E. faecalis</i>, <i>E. faecium</i>, and <i>Listeria</i> in prospectively collected monomicrobial blood cultures by Verigene BC-GP (<i>n = </i>1,157).
a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p
Difference in time to final identification and antimicrobial susceptibility report (<i>n = </i>107).
a<p>Difference in time between BC-GP result and final culture-based identification and susceptibility results.</p>b<p>Includes 26 <i>S. epidermidis</i>.</p>c<p>Three cultures reported as “Not Detected” by BC-GP.</p>d<p>Based on eight cultures that met criteria for full identification and susceptibility testing. Average time to “CoNS” identification only was 27.5 h (range 14 h to 48 h) earlier using BC-GP, <i>n = </i>35.</p>e<p>One culture reported as <i>S. pneumoniae</i> by BC-GP.</p>f<p>Based on six cultures that met criteria for full identification and susceptibility testing.</p>g<p>Culture reported as “<i>Staphylococcus</i> spp.” by BC-GP.</p
Detection of <i>Staphylococcus</i> spp. in prospectively collected monomicrobial blood cultures by Verigene BC-GP (<i>n = </i>1,157).
a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p>c<p>Nucleic acid sequencing confirmed the identification of <i>S. epidermidis</i> in five of ten cultures.</p>d<p>Nucleic acid sequencing confirmed the identification of <i>S. epidermidis</i> in six of ten cultures.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p
Detection of Gram positive identification and resistance determinants from contrived blood cultures by Verigene BC-GP (<i>n = </i>387).
a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p>c<p><i>mecA</i> is reported only for cultures with positive <i>S. aureus</i> or <i>S. epidermidis</i> species targets.</p>d<p><i>vanA</i> and <i>vanB</i> are reported only for cultures with positive <i>E. feacium</i> or <i>E. faecalis</i> targets.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p
Detection of Gram-positive identification and resistance determinants from polymicrobial blood cultures by Verigene BC-GP (<i>n = </i>95).
a<p>Coagulase negative <i>Staphylococcus</i> species, not including <i>S. epidermidis</i> and <i>S. lugdunensis</i>.</p>b<p>Contrived culture.</p>c<p><i>E. faecalis</i> or <i>E. faecium</i>.</p>d<p><i>Gram-negative rod</i>.</p>e<p><i>Bacillus</i> spp. not <i>anthracis</i>.</p
Detection of resistance determinants <i>mecA, vanA, and vanB</i> in prospectively collected monomicrobial blood cultures by Verigene BC-GP (<i>n = </i>599 <i>S. aureus/S. epidermidis</i>, <i>n = </i>81 <i>E. faecalis/E. faecium</i>).
a<p>Total number of cultures with reported result.</p>b<p>95% confidence interval.</p>c<p><i>mecA</i> is reported only for cultures with positive <i>S. aureus</i> or <i>S. epidermidis</i> species targets. Inferred based upon resistance to cefoxitin using disk diffusion method.</p>d<p><i>mecA</i> specific PCR was positive for eight of 14 isolates. Final specificity of 97.5%.</p>e<p><i>mecA</i> specific PCR was negative for four of five isolates. Final sensitivity of 99.7%.</p>f<p>vanA and vanB are reported only for cultures with positive <i>E. feacium</i> or <i>E. faecalis</i> targets.</p>g<p><i>vanB</i> was not detected in any prospectively collected cultures.</p><p>FN, false negative; FP, false positive; TN, true negative; TP, true positive.</p
Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay
Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; bla(CTX-M), 98.9%; bla(KPC), 100%; bla(NDM), 96.2%; bla(OXA), 94.3%; bla(VIM), 100%; and bla(IMP), 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity