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Yonatan H Grad - One of the best experts on this subject based on the ideXlab platform.

  • rna polymerase mutations cause Cephalosporin Resistance in clinical neisseria gonorrhoeae isolates
    eLife, 2020
    Co-Authors: Samantha G Palace, Yi Wang, Daniel H F Rubin, Michael Welsh, Suzanne Walker, Yonatan H Grad, Tatum D Mortimer, Kevin Cole, David W Eyre
    Abstract:

    Increasing Neisseria gonorrhoeae Resistance to ceftriaxone, the last antibiotic recommended for empiric gonorrhea treatment, poses an urgent public health threat. However, the genetic basis of reduced susceptibility to ceftriaxone is not completely understood: while most ceftriaxone Resistance in clinical isolates is caused by target site mutations in penA, some isolates lack these mutations. We show that penA-independent ceftriaxone Resistance has evolved multiple times through distinct mutations in rpoB and rpoD. We identify five mutations in these genes that each increase Resistance to ceftriaxone, including one mutation that arose independently in two lineages, and show that clinical isolates from multiple lineages are a single nucleotide change from ceftriaxone Resistance. These RNA polymerase mutations cause large-scale transcriptional changes without altering susceptibility to other antibiotics, reducing growth rate, or deranging cell morphology. These results underscore the unexpected diversity of pathways to Resistance and the importance of continued surveillance for novel Resistance mutations.

  • o04 1 novel pathway to ceftriaxone Resistance in clinical isolates of n gonorrhoeae via point mutations in the rna polymerase
    Sexually Transmitted Infections, 2019
    Co-Authors: Samantha G Palace, Yi Wang, Daniel B Rubin, Yonatan H Grad
    Abstract:

    Background Widespread antimicrobial Resistance in Neisseria gonorrhoeae has limited the effective treatment options. Cephalosporins remain one of the few classes of antibiotics recommended for gonococcal infections, but reduced susceptibility to the third-generation Cephalosporins, including ceftriaxone, has emerged. Most reduced susceptibility to ceftriaxone is caused by an alternative penA(PBP2) allele. However, the isolates with the among the highest level Cephalosporin Resistance identified in the US lack this allele and other penA Resistance mutations, raising the possibility of Cephalosporin Resistance not mediated directly through penA. Methods To identify the genetic basis of Resistance in these isolates, we employed an undirected transformation strategy, and used molecular microbiology and genetics methods to investigate the mechanism of Resistance. Results Here, we show that Resistance to ESCs has arisen in clinical isolates multiple times through distinct mutations in the RNA polymerase components rpoB and rpoD. The Resistance caused by these changes is not a general tolerance response: these mutations neither changed the growth rate in vitronor altered susceptibility to other classes of antibiotics (including penicillin). These mutations result in large variations in transcription, including in genes coding for penicillin binding proteins (increase in PBP1, decrease in PBPs 3 and 4) and pilus pore. We show that increases in PBP1 protein levels contribute to the rise in CRO MIC, likely through replacement of inhibited PBP2 activity, though other factors are needed to recapitulate the Resistance seen in the clinical isolates with rpoB and rpoD mutations. Conclusion Pathways to extended spectrum Cephalosporin Resistance do not require alterations to penA (PBP2) and can arise through mutations in components of the RNA polymerase holoenzyme. Additional pathways to Cephalosporin Resistance in goncooccus remain to be identified. These findings have implications for the development of molecular diagnostics and for understanding the mechanistic basis of Cephalosporin Resistance. Disclosure No significant relationships.

  • rna polymerase mutations cause Cephalosporin Resistance in clinical neisseria gonorrhoeae isolates
    bioRxiv, 2019
    Co-Authors: Samantha G Palace, Yi Wang, Daniel H F Rubin, Michael Welsh, Suzanne Walker, Yonatan H Grad
    Abstract:

    Abstract The rising incidence of Neisseria gonorrhoeae infection and antimicrobial Resistance imperils effective therapy for gonococcal infections 1–4. Current first-line therapy for gonorrhea relies on ceftriaxone (CRO), an extended spectrum Cephalosporin (ESC), as the backbone of treatment. Dual therapy with azithromycin was intended to delay the emergence of Resistance, but rising azithromycin Resistance rates have been reported 2, 5, resulting in revision of United Kingdom treatment guidelines to CRO monotherapy 6. With no clear next-line agent, gonococcal Resistance to ESCs is a problem of paramount clinical importance, underscored by recent reports of treatment failure and global spread of a multidrug-resistant strain 4, 7. Reduced susceptibility to ceftriaxone (CRORS) is most commonly associated with interspecies mosaic and other alleles of penA (PBP2) 8–10, the primary target of ESCs 8, 11. However, collections of clinical specimens often include isolates with high-level reduced susceptibility to ESCs (ESCRS) that is not attributable to penA variation 3, 12, 13. Here, we describe the genetic basis of reduced susceptibility in three such isolates collected in the United States. Each of these isolates has a unique mutation in the RNA polymerase holoenzyme that causes phenotypic CRORS when introduced into susceptible strains. We demonstrate that other clinical isolates can develop high-level ESCRS via a single nucleotide change in RNA polymerase. This result has important implications regarding the biology underlying Cephalosporin Resistance, the potential for de novo evolution of Resistance under Cephalosporin monotherapy, and the accuracy of sequence-based diagnostics.

Hidemasa Izumiya - One of the best experts on this subject based on the ideXlab platform.

Christopher J Kristich - One of the best experts on this subject based on the ideXlab platform.

  • Multiple Low-Reactivity Class B Penicillin-Binding Proteins Are Required for Cephalosporin Resistance in Enterococci.
    Antimicrobial agents and chemotherapy, 2020
    Co-Authors: Dušanka Djorić, Jaime L. Little, Christopher J Kristich
    Abstract:

    Enterococcus faecalis and Enterococcus faecium are commensals of the gastrointestinal tract of most terrestrial organisms, including humans, and are major causes of health care-associated infections. Such infections are difficult or impossible to treat, as the enterococcal strains responsible are often resistant to multiple antibiotics. One intrinsic Resistance trait that is conserved among E. faecalis and E. faecium is Cephalosporin Resistance, and prior exposure to Cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection. Cephalosporins inhibit peptidoglycan biosynthesis by acylating the active-site serine of penicillin-binding proteins (PBPs) to prevent the PBPs from catalyzing cross-linking during peptidoglycan synthesis. For decades, a specific PBP (known as Pbp4 or Pbp5) that exhibits low reactivity toward Cephalosporins has been thought to be the primary PBP required for Cephalosporin Resistance. We analyzed other PBPs and report that in both E. faecalis and E. faecium, a second PBP, PbpA(2b), is also required for Resistance; notably, the Cephalosporin ceftriaxone exhibits a lethal effect on the ΔpbpA mutant. Strikingly, PbpA(2b) exhibits low intrinsic reactivity with Cephalosporins in vivo and in vitro Unlike the Δpbp5 mutant, the ΔpbpA mutant exhibits a variety of phenotypic defects in growth kinetics, cell wall integrity, and cellular morphology, indicating that PbpA(2b) and Pbp5(4) are not functionally redundant and that PbpA(2b) plays a more central role in peptidoglycan synthesis. Collectively, our results shift the current understanding of enterococcal Cephalosporin Resistance and suggest a model in which PbpA(2b) and Pbp5(4) cooperate to coordinately mediate peptidoglycan cross-linking in the presence of Cephalosporins.

  • Extracellular SalB Contributes to Intrinsic Cephalosporin Resistance and Cell Envelope Integrity in Enterococcus faecalis.
    Journal of bacteriology, 2017
    Co-Authors: Dušanka Djorić, Christopher J Kristich
    Abstract:

    Enterococci are major causes of hospital-acquired infections. Intrinsic Resistance to Cephalosporins is a universal trait among clinically relevant enterococci. Cephalosporin Resistance enables enterococci to proliferate to high densities in the intestines of patients undergoing Cephalosporin treatment, a precursor to the emergence of infection. However, the genetic and biochemical mechanisms of intrinsic Cephalosporin Resistance in enterococci are not well understood. A two-component signal transduction system, CroR/S, is required for Cephalosporin Resistance in enterococci. Although the CroR/S regulon is not well defined, one gene reported to be CroR dependent in Enterococcus faecalis JH2-2 encodes an extracellular putative peptidoglycan hydrolase, SalB. To test the hypothesis that SalB is responsible for CroR-dependent Cephalosporin Resistance, we examined ΔsalB mutants in multiple genetic lineages of E. faecalis, revealing that SalB is required not only for intrinsic Cephalosporin Resistance but also for maintenance of cell envelope integrity in the absence of antibiotic stress. The N-terminal signal sequence is necessary for SalB secretion, and secretion is required for SalB to promote Cephalosporin Resistance. Functional dissection revealed that the C-terminal SCP domain of SalB is essential for biological activity and identified three residues within the SCP domain that are required for the stability and function of SalB. Additionally, we found that in contrast to what is seen in E. faecalis JH2-2, SalB is not regulated by the CroR/S two-component system in E. faecalis OG1, suggesting diversity in the CroR/S regulon among distinct lineages of E. faecalis IMPORTANCE Resistance to Cephalosporins is universal among clinically relevant enterococci, enabling enterococcal proliferation to high densities in the intestines of patients undergoing Cephalosporin treatment, a precursor to the emergence of infection. Disabling Cephalosporin Resistance could therefore reduce the incidence of enterococcal infections. However, the genetic and biochemical mechanisms of Cephalosporin Resistance are not well understood. The significance of this work is the identification of a novel extracellular factor (SalB) that promotes Cephalosporin Resistance in E. faecalis, which could potentially serve as a target for therapeutics that impair enterococcal Cephalosporin Resistance. Additionally, our work highlights the importance of the C-terminal SCP domain of SalB, including several conserved residues within the SCP domain, for the ability of SalB to promote Cephalosporin Resistance.

  • Structure and Dimerization of IreB, a Negative Regulator of Cephalosporin Resistance in Enterococcus faecalis.
    Journal of molecular biology, 2017
    Co-Authors: Cherisse L. Hall, Jessica S. Hoff, Betsy L. Lytle, Davin R. Jensen, Francis C. Peterson, Brian F. Volkman, Christopher J Kristich
    Abstract:

    Enterococcus faecalis, a leading cause of hospital-acquired infections, exhibits intrinsic Resistance to most Cephalosporins, which are antibiotics in the beta-lactam family that target cell-wall biosynthesis. A comprehensive understanding of the underlying genetic and biochemical mechanisms of Cephalosporin Resistance in E. faecalis is lacking. We previously determined that a transmembrane serine/threonine kinase (IreK) and its cognate phosphatase (IreP) reciprocally regulate Cephalosporin Resistance in E. faecalis, dependent on the kinase activity of IreK. Other than IreK itself, thus far the only known substrate for reversible phosphorylation by IreK and IreP is IreB, a small protein of unknown function that is well conserved in low-GC Gram-positive bacteria. We previously showed that IreB acts as a negative regulator of Cephalosporin Resistance in E. faecalis. However, the biochemical mechanism by which IreB modulates Cephalosporin Resistance remains unknown. As a next step toward an understanding of the mechanism by which IreB regulates Resistance, we initiated a structure-function study on IreB. The NMR solution structure of IreB was determined, revealing that IreB adopts a unique fold and forms a dimer in vitro. Dimerization of IreB was confirmed in vivo. Substitutions at the dimer interface impaired IreB function and stability in vivo, indicating that dimerization is functionally important for the biological activity of IreB. Hence, these studies provide new insights into the structure and function of a widely conserved protein of unknown function that is an important regulator of antimicrobial Resistance in E. faecalis.

  • functional dissection of the crors two component system required for Resistance to cell wall stressors in enterococcus faecalis
    Journal of Bacteriology, 2016
    Co-Authors: Stephanie L Kellogg, Christopher J Kristich
    Abstract:

    ABSTRACT Bacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogen Enterococcus faecalis utilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate Resistance to cell wall stresses such as Cephalosporin antibiotics, but the molecular details by which CroRS promotes Cephalosporin Resistance have not been elucidated. Here, we analyzed mutants of E. faecalis carrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives Resistance to Cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylation in vivo . Deletion of croS in various lineages of E. faecalis revealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded by OG1RF_12162 ; here called cisS ). Further analysis of this TCS network revealed that both systems respond to cell wall stress. IMPORTANCE TCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogen Enterococcus faecalis utilizes the CroRS TCS to mediate Resistance to cell wall stresses, including clinically relevant antibiotics such as Cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and Cephalosporin Resistance in E. faecalis cells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and Cephalosporin Resistance in E. faecalis.

  • Thymidylate Limitation Potentiates Cephalosporin Activity toward Enterococci via an Exopolysaccharide-Based Mechanism.
    ACS chemical biology, 2016
    Co-Authors: Jessica S. Hoff, Christopher J Kristich
    Abstract:

    Multidrug resistant enterococci are major causes of nosocomial infections. Prior therapy with Cephalosporins increases the risk of developing an enterococcal infection due to the intrinsic Resistance of enterococci to these antibiotics. While progress has been made toward understanding the genetic and biochemical mechanisms of Cephalosporin Resistance, available data indicate that as-yet-unidentified Resistance factors must exist. Here, we describe results of a screen to identify small molecules capable of sensitizing enterococci to broad-spectrum Cephalosporins. We found that both Enterococcus faecalis and Enterococcus faecium were sensitized to broad and expanded-spectrum Cephalosporins when thymidylate production was impaired, whether by direct inhibition of thymidylate synthase, or by limiting production of cofactors required for its activity. Cephalosporin potentiation is the result of altered exopolysaccharide production due to reduced dTDP-glucose synthesis. Hence, exopolysaccharide production is a previously undescribed contributor to the intrinsic Cephalosporin Resistance of enterococci and serves as a new target for antienterococcal therapeutics.

Sheng Chen - One of the best experts on this subject based on the ideXlab platform.

  • characterization of an inca c multidrug Resistance plasmid in vibrio alginolyticus
    Antimicrobial Agents and Chemotherapy, 2016
    Co-Authors: Dachuan Lin, Yuanjie Zhou, Qiong Ding, Edward Chan, Wen Yao, Sheng Chen
    Abstract:

    Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their Cephalosporin Resistance. The complete sequence of a multidrug Resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae.

  • Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus.
    Antimicrobial agents and chemotherapy, 2016
    Co-Authors: Dachuan Lin, Yuanjie Zhou, Qiong Ding, Edward Chan, Wen Yao, Sheng Chen
    Abstract:

    Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their Cephalosporin Resistance. The complete sequence of a multidrug Resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae.

Masatomo Morita - One of the best experts on this subject based on the ideXlab platform.