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  • American Society for Microbiology  (9)
  • 1
    Online Resource
    Online Resource
    Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection
    American Society for Microbiology ; 2019
    In:  mBio Vol. 10, No. 5 ( 2019-10-29)
    Details
    In: mBio, American Society for Microbiology, Vol. 10, No. 5 ( 2019-10-29)
    Abstract: Strains of Pseudomonas aeruginosa with deficiencies in DNA mismatch repair have been studied in the context of chronic infection, where elevated mutational rates (“hypermutation”) may facilitate the acquisition of antimicrobial resistance. Whether P. aeruginosa hypermutation can also play an adaptive role in the more dynamic context of acute infection remains unclear. In this work, we demonstrate that evolved mismatch repair deficiencies may be exploited by P. aeruginosa to facilitate rapid acquisition of antimicrobial resistance in acute infection, and we directly document rapid clonal succession by such a hypermutating lineage in a patient. Whole-genome sequencing (WGS) was performed on nine serially cultured blood and respiratory isolates from a patient in whom ceftazidime-avibactam (CZA) resistance emerged in vivo over the course of days. The CZA-resistant clone was differentiated by 14 mutations, including a gain-of-function G183D substitution in the PDC-5 chromosomal AmpC cephalosporinase conferring CZA resistance. This lineage also contained a substitution (R656H) at a conserved position in the ATPase domain of the MutS mismatch repair (MMR) protein, and elevated mutational rates were confirmed by mutational accumulation experiments with WGS of evolved lineages in conjunction with rifampin resistance assays. To test whether MMR-deficient hypermutation could facilitate rapid acquisition of CZA resistance, in vitro adaptive evolution experiments were performed with a mutS -deficient strain. These experiments demonstrated rapid hypermutation-facilitated acquisition of CZA resistance compared with the isogenic wild-type strain. Our results suggest a possibly underappreciated role for evolved MMR deficiency in facilitating rapid adaptive evolution of P. aeruginosa in the context of acute infection. IMPORTANCE Antimicrobial resistance in bacteria represents one of the most consequential problems in modern medicine, and its emergence and spread threaten to compromise central advances in the treatment of infectious diseases. Ceftazidime-avibactam (CZA) belongs to a new class of broad-spectrum beta-lactam/beta-lactamase inhibitor combinations designed to treat infections caused by multidrug-resistant bacteria. Understanding the emergence of resistance to this important new drug class is of critical importance. In this work, we demonstrate that evolved mismatch repair deficiency in P. aeruginosa , an important pathogen responsible for significant morbidity and mortality among hospitalized patients, may facilitate rapid acquisition of resistance to CZA in the context of acute infection. These findings are relevant for both diagnosis and treatment of antimicrobial resistance emerging in acute infection in the hypermutator background and additionally have implications for the emergence of more virulent phenotypes.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.01822-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 2557172-2
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  • 2
    Online Resource
    Online Resource
    Manual Reading of Sensititre Broth Microdilution System Panels Improves Accuracy of Susceptibility Reporting for Polymyxin Antibiotics
    American Society for Microbiology ; 2021
    In:  Journal of Clinical Microbiology Vol. 59, No. 9 ( 2021-08-18)
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 59, No. 9 ( 2021-08-18)
    Abstract: Accurate and reproducible antimicrobial susceptibility testing (AST) of polymyxin antibiotics is critical, as these drugs are last-line therapeutic options for the treatment of multidrug-resistant Gram-negative bacterial infections. However, polymyxin AST in the routine laboratory remains challenging. In this study, we evaluated the performance of an automated broth microdilution (BMD) system (Sensititre, ThermoFisher) compared to that of agar dilution (AD) for colistin and polymyxin B AST of 129 Enterobacterales , Pseudomonas aeruginosa , and Acinetobacter baumannii complex clinical isolates. MICs derived from the Sensititre instrument based on two operator comparisons demonstrated overall categorical agreement (CA) of 86% and 89% compared to AD for colistin and 89% and 92% compared to AD for polymyxin B. However, error rates were higher than recommended by CLSI. Manual inspection of microdilution wells revealed microbial growth and skip wells which were erroneously interpreted by the Aris 2X instrument. Using manually interpreted BMD MICs read by two operators increased the overall categorical agreements to 88% and 95% compared to AD for colistin and 92% and 96% compared to AD for polymyxin B. Laboratories choosing to use the Sensititre platform for polymyxin AST should consider manual evaluation of wells as part of their algorithm.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.00332-21
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Detection and Whole-Genome Sequencing of Carbapenemase-Producing Aeromonas hydrophila Isolates from Routine Perirectal Surveillance Culture
    American Society for Microbiology ; 2016
    In:  Journal of Clinical Microbiology Vol. 54, No. 4 ( 2016-04), p. 1167-1170
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 54, No. 4 ( 2016-04), p. 1167-1170
    Abstract: Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the bla KPC-2 gene in one isolate prompted inclusion of non- Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.03229-15
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 1498353-9
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  • 4
    Online Resource
    Online Resource
    Reply to “Tn 4401 Carrying bla KPC Is Inserted within Another Insertion in pKpQIL and Related Plasmids”
    American Society for Microbiology ; 2014
    In:  Journal of Clinical Microbiology Vol. 52, No. 12 ( 2014-12), p. 4450-4450
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 52, No. 12 ( 2014-12), p. 4450-4450
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.02614-14
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 1498353-9
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  • 5
    Online Resource
    Online Resource
    A Rapid Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant Enterobacteriaceae
    American Society for Microbiology ; 2014
    In:  Journal of Clinical Microbiology Vol. 52, No. 8 ( 2014-08), p. 2804-2812
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 52, No. 8 ( 2014-08), p. 2804-2812
    Abstract: Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the bla KPC carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼11,109-Da MS peak corresponding to a gene product of the bla KPC pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of bla KPC -negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the bla KPC Tn 4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla KPC Tn 4401 -containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.00694-14
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    Clinical Performance of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Detection of Certain bla KPC -Containing Plasmids
    American Society for Microbiology ; 2016
    In:  Journal of Clinical Microbiology Vol. 54, No. 1 ( 2016-01), p. 35-42
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 54, No. 1 ( 2016-01), p. 35-42
    Abstract: Rapid detection of bla KPC -containing organisms can significantly impact infection control and clinical practices, as well as therapeutic choices. Current molecular and phenotypic methods to detect these organisms, however, require additional testing beyond routine organism identification. In this study, we evaluated the clinical performance of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to detect pKpQIL_p019 (p019)—an ∼11,109-Da protein associated with certain bla KPC -containing plasmids that was previously shown to successfully track a clonal outbreak of bla KPC -pKpQIL- Klebsiella pneumoniae in a proof-of-principle study (A. F. Lau, H. Wang, R. A. Weingarten, S. K. Drake, A. F. Suffredini, M. K. Garfield, Y. Chen, M. Gucek, J. H. Youn, F. Stock, H. Tso, J. DeLeo, J. J. Cimino, K. M. Frank, and J. P. Dekker, J Clin Microbiol 52:2804–2812, 2014, http://dx.doi.org/10.1128/JCM.00694-14 ). PCR for the p019 gene was used as the reference method. Here, blind analysis of 140 characterized Enterobacteriaceae isolates using two protein extraction methods (plate extraction and tube extraction) and two peak detection methods (manual and automated) showed sensitivities and specificities ranging from 96% to 100% and from 95% to 100%, respectively (2,520 spectra analyzed). Feasible laboratory implementation methods (plate extraction and automated analysis) demonstrated 96% sensitivity and 99% specificity. All p019-positive isolates ( n = 26) contained bla KPC and were carbapenem resistant. Retrospective analysis of an additional 720 clinical Enterobacteriaceae spectra found an ∼11,109-Da signal in nine spectra (1.3%), including seven from p019-containing, carbapenem-resistant isolates (positive predictive value [PPV], 78%). Instrument tuning had a significant effect on assay sensitivity, highlighting important factors that must be considered as MALDI-TOF MS moves into applications beyond microbial identification. Using a large blind clinical data set, we have shown that spectra acquired for routine organism identification can also be analyzed automatically in real time at high throughput, at no additional expense to the laboratory, to enable rapid detection of potentially bla KPC -containing carbapenem-resistant isolates, providing early and clinically actionable results.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.01643-15
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    Frequent and Variable Cytotoxic-T-Lymphocyte Escape-Associated Fitness Costs in the Human Immunodeficiency Virus Type 1 Subtype B Gag Proteins
    American Society for Microbiology ; 2013
    In:  Journal of Virology Vol. 87, No. 7 ( 2013-04), p. 3952-3965
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 87, No. 7 ( 2013-04), p. 3952-3965
    Abstract: Cytotoxic-T-lymphocyte (CTL) escape mutations undermine the durability of effective human immunodeficiency virus type 1 (HIV-1)-specific CD8 + T cell responses. The rate of CTL escape from a given response is largely governed by the net of all escape-associated viral fitness costs and benefits. The observation that CTL escape mutations can carry an associated fitness cost in terms of reduced virus replication capacity (RC) suggests a fitness cost-benefit trade-off that could delay CTL escape and thereby prolong CD8 response effectiveness. However, our understanding of this potential fitness trade-off is limited by the small number of CTL escape mutations for which a fitness cost has been quantified. Here, we quantified the fitness cost of the 29 most common HIV-1B Gag CTL escape mutations using an in vitro RC assay. The majority (20/29) of mutations reduced RC by more than the benchmark M184V antiretroviral drug resistance mutation, with impacts ranging from 8% to 69%. Notably, the reduction in RC was significantly greater for CTL escape mutations associated with protective HLA class I alleles than for those associated with nonprotective alleles. To speed the future evaluation of CTL escape costs, we also developed an in silico approach for inferring the relative impact of a mutation on RC based on its computed impact on protein thermodynamic stability. These data illustrate that the magnitude of CTL escape-associated fitness costs, and thus the barrier to CTL escape, varies widely even in the conserved Gag proteins and suggest that differential escape costs may contribute to the relative efficacy of CD8 responses.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.03233-12
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2013
    detail.hit.zdb_id: 1495529-5
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  • 8
    Online Resource
    Online Resource
    An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion
    American Society for Microbiology ; 2019
    In:  mBio Vol. 10, No. 5 ( 2019-10-29)
    Details
    In: mBio, American Society for Microbiology, Vol. 10, No. 5 ( 2019-10-29)
    Abstract: The human fungal commensal Candida albicans can become a serious opportunistic pathogen in immunocompromised hosts. The C. albicans cell adhesion protein Als1p is a highly expressed member of a large family of paralogous adhesins. Als1p can mediate binding to epithelial and endothelial cells, is upregulated in infections, and is important for biofilm formation. Als1p includes an amyloid-forming sequence at amino acids 325 to 331, identical to the sequence in the paralogs Als5p and Als3p. Therefore, we mutated Val326 to test whether this sequence is important for activity. Wild-type Als1p (Als1p WT ) and Als1p with the V326N mutation (Als1p V326N ) were expressed at similar levels in a Saccharomyces cerevisiae surface display model. Als1p V326N cells adhered to bovine serum albumin (BSA)-coated beads similarly to Als1p WT cells. However, cells displaying Als1p V326N showed visibly smaller aggregates and did not fluoresce in the presence of the amyloid-binding dye Thioflavin-T. A new analysis tool for single-molecule force spectroscopy-derived surface mapping showed that statistically significant force-dependent Als1p clustering occurred in Als1p WT cells but was absent in Als1p V326N cells. In single-cell force spectroscopy experiments, strong cell-cell adhesion was dependent on an intact amyloid core sequence on both interacting cells. Thus, the major adhesin Als1p interacts through amyloid-like β-aggregation to cluster adhesin molecules in cis on the cell surface as well as in trans to form cell-cell bonds. IMPORTANCE Microbial cell surface adhesins control essential processes such as adhesion, colonization, and biofilm formation. In the opportunistic fungal pathogen Candida albicans , the a gglutinin- l ike s equence ( ALS ) gene family encodes eight cell surface glycoproteins that mediate adherence to biotic and abiotic surfaces and cell-cell aggregation. Als proteins are critical for commensalism and virulence. Their activities include attachment and invasion of endothelial and epithelial cells, morphogenesis, and formation of biofilms on host tissue and indwelling medical catheters. At the molecular level, Als5p-mediated cell-cell aggregation is dependent on the formation of amyloid-like nanodomains between Als5p-expressing cells. A single-site mutation to valine 326 abolishes cellular aggregation and amyloid formation. Our results show that the binding characteristics of Als1p follow a mechanistic model similar to Als5p, despite its differential expression and biological roles.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.01766-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 2557172-2
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  • 9
    Online Resource
    Online Resource
    Multicenter Comparison of Nucleic Acid Amplification Tests for the Diagnosis of Rectal and Oropharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae Infections
    American Society for Microbiology ; 2022
    In:  Journal of Clinical Microbiology Vol. 60, No. 1 ( 2022-01-19)
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 60, No. 1 ( 2022-01-19)
    Abstract: Research using nucleic acid amplification tests (NAATs) have repeatedly found rectal and oropharyngeal infections with Chlamydia trachomatis and Neisseria gonorrhoeae to be common and potentially more difficult to treat than genital infections. Unfortunately, public health and patient care efforts have been hampered by the lack of FDA-cleared NAATs with claims for anorectal or oropharyngeal samples. At the time of the initiation of this study, no commercially available assays had these claims. We formed a novel partnership among academic institutions and diagnostic manufacturers to address this public health need. From May 2018 through August 2019, we recruited 1108 women, 1256 men, and 26 transgender persons each of whom provided 3 anal and 3 oropharyngeal swab specimens. The 3 anal swabs were pooled into a single transport tube as were the 3 oropharyngeal swabs. The performance of each of three study assays was estimated by comparison to the composite result and relative to one another. Percent positivity for chlamydia was 5.9 and 1.2% from anal and oropharyngeal specimens, respectively, compared to 4.2 and 4.1% for gonorrhea. Sensitivity for chlamydia detection ranged from 81.0 to 95.1% and 82.8 to 100% for anal and oropharyngeal specimens, respectively. Gonorrhea sensitivity ranged from 85.9 to 99.0% and 74.0 to 100% for anal and oropharyngeal samples, respectively. Specificity estimates were ≥ 98.9% for all assays, organisms, and sample types. Although there was heterogeneity between sensitivity estimates, these assays offer better ability to detect extragenital infections than culture and potential solutions for providing appropriate sexual health care for populations in which these infections are of concern.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.01363-21
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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