Mechanisms of linezolid resistance among coagulase-negative staphylococci determined by whole-genome sequencing.

UnlabelledLinezolid resistance is uncommon among staphylococci, but approximately 2% of clinical isolates of coagulase-negative staphylococci (CoNS) may exhibit resistance to linezolid (MIC, ≥8 µg/ml). We performed whole-genome sequencing (WGS) to characterize the resistance mechanisms and genetic backgrounds of 28 linezolid-resistant CoNS (21 Staphylococcus epidermidis isolates and 7 Staphylococcus haemolyticus isolates) obtained from blood cultures at a large teaching health system in California between 2007 and 2012. The following well-characterized mutations associated with linezolid resistance were identified in the 23S rRNA: G2576U, G2447U, and U2504A, along with the mutation C2534U. Mutations in the L3 and L4 riboproteins, at sites previously associated with linezolid resistance, were also identified in 20 isolates. The majority of isolates harbored more than one mutation in the 23S rRNA and L3 and L4 genes. In addition, the cfr methylase gene was found in almost half (48%) of S. epidermidis isolates. cfr had been only rarely identified in staphylococci in the United States prior to this study. Isolates of the same sequence type were identified with unique mutations associated with linezolid resistance, suggesting independent acquisition of linezolid resistance in each isolate.ImportanceLinezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria

Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay

<p>Abstract</p> <p>Background</p> <p>The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite <it>Plasmodium falciparum</it>, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In <it>P. falciparum</it>, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.</p> <p>Results</p> <p>We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity <it>in vitro</it>. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme.</p> <p>Conclusion</p> <p>The identification of amodiaquine as an inhibitor of PfPMT <it>in vitro </it>and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.</p

Role of Plasmodium falciparum Phosphoethanolamine Methyltransferase in Parasite Development and Differentiation

Plasmodium falciparum is an intra-erythrocytic parasite that causes the human disease, malaria. Current antimalarial therapies target the asexual intra-erythrocytic stages of the parasite, preventing clinical manifestations of the disease, but failing to block gametocyte differentiation a key stage for transmission of the disease. The development of drug resistance to most of the currently available antimalarials has created an urgent need for the identification of novel chemical compounds, and for the discovery of new biological pathways in the parasite to be used as targets in antimalarial therapy. Phospholipid synthesis pathways in the parasite have shown to be promising targets for antimalarial drug development. P. falciparum phosphoethanolamine methyltransferase, PfPMT is an enzyme that is unique to human malaria parasites, nematodes, and plants, but absent in humans. PfPMT catalyzes the methylation of phosphoethanolamine to phosphocholine, which feeds into the CDP-choline pathway synthesizing the essential phospholipid, phosphatidylcholine. ^ Genetic disruption of the Pfpmt gene results in a severe growth defect in asexual parasite development. But more importantly we found that P. falciparum parasites lacking PIPMT were unable to undergo gametocyte differentiation. This finding suggests that PfPMT is essential for gametocyte development and could be a target for the development of transmission blocking drugs.^ An in vitro enzyme-coupled spectrophotometeric assay was developed for the identification of inhibitors of PfPMT activity. Surprisingly we found the known antimalarial drug amodiaquine to be a strong inhibitor of PfPMT activity. Using this assay to screen a small chemical compound library, we found eleven compounds to inhibit PfPMT activity and asexual parasite replication in the low micromolar range. Two of these compounds were also found to inhibit gametocyte differentiation.^ With the current status of today\u27s antimalarials, there is a great need for new compounds that can inhibit both infection and transmission. Our analysis of PfPMT and its role in asexual development and sexual differentiation have led us to conclude that PfPMT is a valuable drug target. Additionally we\u27ve identified novel compounds that could be used as leads in the development of new antimalarial drugs.

Frequency of Susceptibility Testing for Patients with Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia

Currently, no standards exist for determining the optimal frequency of repeat antimicrobial susceptibility testing (AST) when an organism is recurrently isolated from the same specimen source. Although testing every 2 to 5 days is thought sufficient, we present three cases of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia where current laboratory protocol for repeating AST every 5 days was inadequate to identify resistant organisms

PG12, a Phospholipid Analog with Potent Antimalarial Activity, Inhibits Plasmodium falciparum CTP:Phosphocholine Cytidylyltransferase Activity

In the human malaria parasite Plasmodium falciparum, the synthesis of the major and essential membrane phospholipid, phosphatidylcholine, occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation (SDPM) pathways, which are fueled by host choline, serine, and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes, P. falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) and choline-phosphate transferase (PfCEPT). We identified a novel class of phospholipid mimetics, which inhibit the growth of P. falciparum as well as Leishmania and Trypanosoma species. Metabolic analyses showed that one of these compounds, PG12, specifically blocks phosphatidylcholine biosynthesis from both the CDP-choline and SDPM pathways via inhibition of PfCCT. In vitro studies using recombinant PfCCT showed a dose-dependent inhibition of the enzyme by PG12. The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo.We thank the World Health Organization, Dr. Foluke Fakorede, and Dr. Reto Brun for the preliminary screening of compounds under the WHO/TDR Training in Tropical Diseases program; A. G. Fernández and J. Casas for discussions; A. Gonzalez- Roura for early synthetic work; and S. Gonzalez and M. Lavigne for technical support.Peer reviewe