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  • 1
    Language: English
    In: Analytical chemistry, 03 February 2015, Vol.87(3), pp.1941-9
    Description: Flow cytometry holds promise to accelerate antibiotic susceptibility determinations; however, without robust multidimensional statistical analysis, general discrimination criteria have remained elusive. In this study, a new statistical method, probability binning signature quadratic form (PB-sQF), was developed and applied to analyze flow cytometric data of bacterial responses to antibiotic exposure. Both sensitive lab strains (Escherichia coli and Pseudomonas aeruginosa) and a multidrug resistant, clinically isolated strain (E. coli) were incubated with the bacteria-targeted dye, maltohexaose-conjugated IR786, and each of many bactericidal or bacteriostatic antibiotics to identify changes induced around corresponding minimum inhibition concentrations (MIC). The antibiotic-induced damages were monitored by flow cytometry after 1-h incubation through forward scatter, side scatter, and fluorescence channels. The 3-dimensional differences between the flow cytometric data of the no-antibiotic treated bacteria and the antibiotic-treated bacteria were characterized by PB-sQF into a 1-dimensional linear distance. A 99% confidence level was established by statistical bootstrapping for each antibiotic-bacteria pair. For the susceptible E. coli strain, statistically significant increments from this 99% confidence level were observed from 1/16x MIC to 1x MIC for all the antibiotics. The same increments were recorded for P. aeruginosa, which has been reported to cause difficulty in flow-based viability tests. For the multidrug resistant E. coli, significant distances from control samples were observed only when an effective antibiotic treatment was utilized. Our results suggest that a rapid and robust antimicrobial susceptibility test (AST) can be constructed by statistically characterizing the differences between sample and control flow cytometric populations, even in a label-free scheme with scattered light alone. These distances vs paired controls coupled with rigorous statistical confidence limits offer a new path toward investigating initial biological responses, screening for drugs, and shortening time to result in antimicrobial sensitivity testing.
    Keywords: Anti-Bacterial Agents -- Pharmacology ; Escherichia Coli -- Drug Effects ; Flow Cytometry -- Methods ; Microbial Sensitivity Tests -- Methods ; Pseudomonas Aeruginosa -- Drug Effects
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 2
    In: Nature, 2013, Vol.497(7448), p.254
    Description: CRISPR/Cas (clustered regularly interspaced palindromic repeats/ CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived frombacteriophages or exogenous plasmids. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innateimmune response aimed at combating pathogens, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts. [PUBLICATION ]
    Keywords: Animals–Genetics ; Female–Immunology ; Gammaproteobacteria–Metabolism ; Gammaproteobacteria–Pathogenicity ; Gammaproteobacteria–Genetics ; Gammaproteobacteria–Immunology ; Genes, Bacterial–Immunology ; Host-Pathogen Interactions–Genetics ; Immune Evasion–Metabolism ; Immunity, Innate–Immunology ; Mice–Metabolism ; Mice, Inbred C57bl–Genetics ; Phylogeny–Genetics ; RNA, Bacterial–Genetics ; RNA, Bacterial–Genetics ; Time Factors–Genetics ; Toll-Like Receptor 2–Genetics ; Toll-Like Receptor 2–Genetics ; Virulence–Genetics ; Proteins ; Mutation ; Bacteria ; Infections ; Binding Sites ; RNA, Bacterial ; Toll-Like Receptor 2;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    In: Nature, 2013, Vol.501(7466), p.262
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 4
    Language: English
    In: BBA - Biomembranes, November 2015, Vol.1848(11), pp.3026-3031
    Description: Antimicrobial peptides (AMPs) play an important role as a host defense against microbial pathogens and are key components of the human innate immune response. frequently colonizes the human nasopharynx as a commensal but also is a worldwide cause of epidemic meningitis and rapidly fatal sepsis. In the human respiratory tract, the only known reservoir of , meningococci are exposed to human endogenous AMPs. Thus, it is not surprising that meningococci have evolved effective mechanisms to confer intrinsic and high levels of resistance to the action of AMPs. This article reviews the current knowledge about AMP resistance mechanisms employed by . Two major resistance mechanisms employed by meningococci are the constitutive modification of the lipid A head groups of lipooligosaccharides by phosphoethanolamine and the active efflux pump mediated excretion of AMPs. Other factors influencing AMP resistance, such as the major porin PorB, the pilin biogenesis apparatus, and capsular polysaccharides, have also been identified. Even with an inherently high intrinsic resistance, several AMP resistance determinants can be further induced upon exposure to AMPs. Many well-characterized AMP resistance mechanisms in other Gram-negative bacteria are not found in meningococci. Thus, utilizes a limited but highly effective set of molecular mechanisms to mediate antimicrobial peptide resistance. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.
    Keywords: Neisseria Meningitidis ; Antimicrobial Resistance ; Efflux Pump ; Phosphoethanolamine Modification of Lipid A ; Chemistry
    ISSN: 0005-2736
    E-ISSN: 1879-2642
    E-ISSN: 18782434
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  • 5
    Language: English
    In: Infection and Immunity, 2012, Vol. 80(2), p.657
    Description: Neisseria meningitidis employs redundant heme acquisition mechanisms, including TonB receptor-dependent and receptor-independent uptakes. The TonB-dependent zinc receptor ZnuD shares significant sequence similarity to HumA, a heme receptor of Moraxella catarrhalis, and contains conserved motifs found in many heme utilization proteins. We present data showing that, when expressed in Escherichia coli, ZnuD allowed heme capture on the cell surface and supported the heme-dependent growth of an E. coli hemA strain. Heme agarose captured ZnuD in enriched outer membrane fractions, and this binding was inhibited by excess free heme, supporting ZnuD's specific interaction with heme. However, no heme utilization defect was detected in the meningococcal znuD mutant, likely due to unknown redundant TonB-independent heme uptake mechanisms. Meningococcal replication within epithelial cells requires a functional TonB, and we found that both the znuD and tonB mutants were defective not only in survival within epithelial cells but also in adherence to and invasion of epithelial cells. Ectopic complementation rescued these phenotypes. Interestingly, while znuD expression was repressed by Zur with zinc as a cofactor, it also was induced by iron in a Zur-independent manner. A specific interaction of meningococcal Fur protein with the znuD promoter was demonstrated by electrophoretic mobility shift assay (EMSA). Thus, the meningococcal ZnuD receptor likely participates in both zinc and heme acquisition, is regulated by both Zur and Fur, and is important for meningococcal interaction with epithelial cells.
    Keywords: Medicine ; Biology;
    ISSN: 1098-5522
    ISSN: 10985522
    ISSN: 00199567
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  • 6
    In: Molecular Microbiology, March 2011, Vol.79(6), pp.1557-1573
    Description: Proper periplasmic disulfide bond formation is important for folding and stability of many secreted and membrane proteins, and is catalysed by three DsbA oxidoreductases in . DsbD provides reducing power to DsbC that shuffles incorrect disulfide bond in misfolded proteins as well as to the periplasmic enzymes that reduce apo‐cytochrome (CcsX) or repair oxidative protein damages (MrsAB). The expression of , but not other genes, is positively regulated by the MisR/S two‐component system. Quantitative real‐time PCR analyses showed significantly reduced expression in all mutants, which was rescued by genetic complementation. The direct and specific interaction of MisR with the upstream region of the promoter was demonstrated by electrophoretic mobility shift assay, and the MisR binding sequences were mapped. Further, the expression of was found to be induced by dithiothrietol (DTT), through the MisR/S regulatory system. Surprisingly, we revealed that inactivation of can only be achieved in a strain carrying an ectopically located , in the double mutant or in the triple mutant, thus DsbD is indispensable for DsbA‐catalysed oxidative protein folding in . The defects of the meningococcal mutant in transformation and resistance to oxidative stress were more severe in the absence of .
    Keywords: Transformation ; Promoters ; Complementation ; Protein Folding ; Oxidative Stress ; Disulfide Bonds ; Polymerase Chain Reaction ; Enzymes ; Oxidoreductase ; Membrane Proteins ; Dithiothreitol ; Electrophoretic Mobility ; Neisseria Meningitidis ; Genetics & Taxonomy;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 7
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 18 April 2017, Vol.114(16), pp.4237-4242
    Description: (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasive meningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, US_NmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of US_NmUC isolates was an IS1301 element in the intergenic region separating the capsule operons and adjacent deletion of and a part of , encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an ∼20-kb sequence near the locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification B-A gene cassette, and strains grow well anaerobically. The cc11 US_NmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the - cassette promoting anaerobic growth.
    Keywords: Is1301 ; Neisseria Meningitidis ; Capsule ; Denitrification ; Meningococcal Urethritis ; Whole Genome Sequencing ; Meningitis, Meningococcal -- Epidemiology ; Neisseria Meningitidis -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 8
    Language: English
    In: Clinical Epidemiology, Annual, 2012, Vol.1, p.237(9)
    Description: The human bacterial pathogen Neisseria meningitidis remains a serious worldwide health threat, but progress is being made toward the control of meningococcal infections. This review summarizes current knowledge of the global epidemiology and the pathophysiology of meningococcal disease, as well as recent advances in prevention by new vaccines. Meningococcal disease patterns and incidence can vary dramatically, both geographically and over time in populations, influenced by differences in invasive meningococcal capsular serogroups and specific genotypes designated as ST clonal complexes. Serogroup A (ST-5, ST-7), B (ST-41/44, ST-32, ST-18, ST-269, ST-8, ST-35), C (ST-11), Y (ST-23, ST-167), W-135 (ST-11) and X (ST-181) meningococci currently cause almost all invasive disease. Serogroups B, C, and Y are responsible for the majority of cases in Europe, the Americas, and Oceania; serogroup A has been associated with the highest incidence (up to 1000 per 100,000 cases) and large outbreaks of meningococcal disease in sub-Saharan Africa and previously Asia; and serogroups W-135 and X have emerged to cause major disease outbreaks in sub-Saharan Africa. Significant declines in meningococcal disease have occurred in the last decade in many developed countries. In part, the decline is related to the introduction of new meningococcal vaccines. Serogroup C polysaccharide-protein conjugate vaccines were introduced over a decade ago, first in the UK in a mass vaccination campaign, and are now widely used; multivalent meningococcal conjugate vaccines containing serogroups A, C, W-135, and/or Y were first used for adolescents in the US in 2005 and have now expanded indications for infants and young children, and a new serogroup A conjugate vaccine has recently been introduced in sub-Saharan Africa. The effectiveness of these conjugate vaccines has been enhanced by the prevention of person-to-person transmission and herd immunity. In addition, progress has been made in serogroup B-specific vaccines based on conserved proteins and outer membrane vesicles. However, continued global surveillance is essential in understanding and predicting the dynamic changes in the epidemiology and biological basis of meningococcal disease and to influence the recommendations for current and future vaccines or other prevention strategies. Keywords: Neisseria meningitidis, meningococcal disease, conjugate vaccines, meningococcal vaccines
    Keywords: Vaccines -- Health Aspects ; Meningitis -- Prevention ; Meningitis -- Health Aspects ; Epidemiology -- Health Aspects
    ISSN: 1179-1349
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Clinical Epidemiology, Annual, 2012, Vol.1, p.237(9)
    Description: The human bacterial pathogen Neisseria meningitidis remains a serious worldwide health threat, but progress is being made toward the control of meningococcal infections. This review summarizes current knowledge of the global epidemiology and the pathophysiology of meningococcal disease, as well as recent advances in prevention by new vaccines. Meningococcal disease patterns and incidence can vary dramatically, both geographically and over time in populations, influenced by differences in invasive meningococcal capsular serogroups and specific genotypes designated as ST clonal complexes. Serogroup A (ST-5, ST-7), B (ST-41/44, ST-32, ST-18, ST-269, ST-8, ST-35), C (ST-11), Y (ST-23, ST-167), W-135 (ST-11) and X (ST-181) meningococci currently cause almost all invasive disease. Serogroups B, C, and Y are responsible for the majority of cases in Europe, the Americas, and Oceania; serogroup A has been associated with the highest incidence (up to 1000 per 100,000 cases) and large outbreaks of meningococcal disease in sub-Saharan Africa and previously Asia; and serogroups W-135 and X have emerged to cause major disease outbreaks in sub-Saharan Africa. Significant declines in meningococcal disease have occurred in the last decade in many developed countries. In part, the decline is related to the introduction of new meningococcal vaccines. Serogroup C polysaccharide-protein conjugate vaccines were introduced over a decade ago, first in the UK in a mass vaccination campaign, and are now widely used; multivalent meningococcal conjugate vaccines containing serogroups A, C, W-135, and/or Y were first used for adolescents in the US in 2005 and have now expanded indications for infants and young children, and a new serogroup A conjugate vaccine has recently been introduced in sub-Saharan Africa. The effectiveness of these conjugate vaccines has been enhanced by the prevention of person-to-person transmission and herd immunity. In addition, progress has been made in serogroup B-specific vaccines based on conserved proteins and outer membrane vesicles. However, continued global surveillance is essential in understanding and predicting the dynamic changes in the epidemiology and biological basis of meningococcal disease and to influence the recommendations for current and future vaccines or other prevention strategies. Keywords: Neisseria meningitidis, meningococcal disease, conjugate vaccines, meningococcal vaccines
    Keywords: Vaccines -- Health Aspects ; Meningitis -- Prevention ; Meningitis -- Health Aspects ; Epidemiology -- Health Aspects
    ISSN: 1179-1349
    Source: Cengage Learning, Inc.
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  • 10
    In: Infection and Immunity, 2010, Vol. 78(3), p.1109
    Description: Outer membrane iron receptors are some of the major surface entities that are critical for meningococcal pathogenesis. The gene encoding the meningococcal hemoglobin receptor, HmbR, is both independently transcribed and transcriptionally linked to the upstream gene hemO, which encodes a heme oxygenase. The MisR/S two-component system was previously determined to regulate hmbR transcription, and its hemO and hmbR regulatory mechanisms were characterized further here. The expression of hemO and hmbR was downregulated in misR/S mutants under both iron-replete and iron-restricted conditions, and the downregulation could be reversed by complementation. No significant changes in expression of other iron receptors were detected, suggesting that the MisR/S system specifically regulates hmbR. When hemoglobin was the sole iron source, growth defects were detected in the mutants. Primer extension analysis identified a promoter upstream of the hemO-associated Correia element (CE) and another promoter at the proximal end of CE, and processed transcripts previously identified for other cotranscribed CEs were also detected, suggesting that there may be posttranscriptional regulation. MisR directly interacts with sequences upstream of the CE and upstream of the hmbR Fur binding site and thus independently regulates hemO and hmbR. Analysis of transcriptional reporters of hemO and hmbR further demonstrated the positive role of the MisR/S system and showed that the transcription of hmbR initiated from hemO was significantly reduced. A comparison of the effects of the misS mutation under iron-replete and iron-depleted conditions suggested that activation by the MisR/S system and iron-mediated repression by Fur act independently. Thus, the expression of hemO and hmbR is coordinately controlled by multiple independent regulatory mechanisms, including the MisR/S two-component system.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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