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Molnupiravir (EIDD-2801/MK-4482), the prodrug of the active antiviral ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931), has activity against a number of RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and seasonal and pandemic influenza viruses. Single and multiple doses of molnupiravir were evaluated in this first-in-human, phase 1, randomized, double-blind, placebo-controlled study in healthy volunteers, which included evaluation of the effect of food on pharmacokinetics.

ABSTRACTCoagulase-negative staphylococci (CoNS) have become the leading cause of bloodstream infections (BSIs) in intensive care units (ICUs), particularly in premature neonates. Vancomycin-intermediate heteroresistant CoNS (hVICoNS) have been identified as sources of BSIs worldwide, and their potential to emerge as significant pathogens in the neonatal ICU (NICU) remains uncertain. This study describes the molecular epidemiology of an outbreak of vancomycin-heteroresistant (hV) Staphylococcus epidermidiscentral-line-associated BSI (CLABSI) in a single tertiary care NICU and compares it to a second tertiary care NICU that had not been associated with an outbreak. Between November 2009 and April 2014, 119 S. epidermidisCLABSIs were identified in two tertiary care NICUs in Quebec, Canada. Decreased vancomycin susceptibility was identified in about 88% of all collected strains using Etest methods. However, discrepancies were found according to the Etest and population analysis profiling–area under the concentration-time curve (PAP-AUC) methods used. All strains were susceptible to linezolid, and a few isolates were nonsusceptible to daptomycin. Great genetic diversity was observed within the collection, with 31 pulsed-field gel electrophoresis (PFGE) patterns identified. The outbreak strains were all determined to be heteroresistant to vancomycin and were polyclonal. The study identified two major clones, PFGE patterns E and G, which were found in both NICUs across the 5-year study period. This suggests the persistence of highly successful clones that are well adapted to the hospital environment. hV S. epidermidisseems more common than currently realized in the NICU, and certain hV S. epidermidisclones can become endemic to the NICU. The reservoirs for these clones remain unknown at this time, and identification of the reservoirs is needed to better understand the impact of hV S. epidermidisin the NICU and to inform infection prevention strategies. In addition, there is a need to investigate and validate hV determination protocols for different species of CoNS.

ABSTRACTCryptococcus gattiiisolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattiiis unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37°C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the pdr11Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattiiclinical isolates should be assessed further in order to identify other potential mechanisms of resistance.

ABSTRACTResiquimod, a Toll-like receptor 7 and 8 agonist, stimulates production of cytokines that promote an antigen-specific T helper type 1 acquired immune response. Animal and phase II human trials showed posttreatment efficacy in reducing recurrent herpes lesion days and/or time to first recurrence. Three phase III randomized, double-blind, vehicle-controlled trials of topical resiquimod to reduce anogenital herpes recurrences were conducted in healthy adults with ≥4 recurrences within the prior year. Participants applied resiquimod 0.01% gel or vehicle gel 2 times per week for 3 weeks to each recurrence for 12 months. Trials 1 and 2 had 2:1 resiquimod-vehicle randomization. Trial 3 had 1:1:1 randomization for resiquimod and 500 mg valacyclovir orally twice daily for 5 days (RESI-VAL), resiquimod and oral placebo (RESI-PLA), and vehicle and oral placebo (VEH-PLA). The median time to first recurrence was similar for resiquimod and vehicle (trial 1, 60 and 56 days, P= 0.7; trial 2, 54 and 48 days, P= 0.47; trial 3, 51 [RESI-VAL], 55 [RESI-PLA], and 44 [VEH-PLA] days, P= not significant [NS]). The median time to healing of initial treated recurrence was longer for resiquimod (trial 1, 18 compared to 10 days, P< 0.001; trial 2, 19 compared to 13 days, P= 0.16; trial 3, 14 [RESI-VAL], 16 [RESI-PLA], and 8 [VEH-PLA] days, P< 0.001). In trials 1 and 2, moderate to severe erythema and erosion/ulceration at the application site were more common in resiquimod recipients. In conclusion, no posttreatment efficacy of resiquimod 0.01% gel was observed. Increased application site reactions and initial recurrence healing time are consistent with resiquimod-induced cytokine effects.

ABSTRACTPseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial biofilm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB, N-mercaptoacetyl-Phe-Tyr-amide (Ki= 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in biofilm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal biofilms, and to eradicate biofilm completely when used in combination with conventional antibiotics.

ABSTRACTCMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC50s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC50s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitrocytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

ABSTRACTThe combination of ST-246 and hexadecyloxypropyl-cidofovir or CMX001 was evaluated for synergistic activity in vitro against vaccinia virus and cowpox virus (CV) and in vivo against CV. In cell culture the combination was highly synergistic against both viruses, and the results suggested that combined treatment with these agents might offer superior efficacy in vivo. For animal models, ST-246 was administered orally with or without CMX001 to mice lethally infected with CV. Treatments began 1, 3, or 6 days postinfection using lower dosages than previously used for single-drug treatment. ST-246 was given at 10, 3, or 1 mg/kg of body weight with or without CMX001 at 3, 1, or 0.3 mg/kg to evaluate potential synergistic interactions. Treatment beginning 6 days post-viral inoculation with ST-246 alone only increased the mean day to death at 10 or 3 mg/kg but had no effect on survival. CMX001 alone also had no effect on survival. When the combination of the two drugs was begun 6 days after viral infection using various dosages of the two, a synergistic reduction in mortality was observed. No evidence of increased toxicity was noted with the combination either in vitro or in vivo. These results indicate that combinations of ST-246 and CMX001 are synergistic both in vitro and in vivo and suggest that combination therapy using ST-246 and CMX001 for treatment of orthopoxvirus disease in humans or animals may provide an additional benefit over the use of the two drugs by themselves.

ABSTRACT9-R-[2-(Phosphonomethoxy)propyl]-adenine (tenofovir) is an acyclic nucleoside phosphonate with antiviral activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). Tenofovir is not orally bioavailable but becomes orally active against HIV-1 infection as the disoproxil ester (tenofovir disoproxil fumarate [Viread]). We have developed an alternative strategy for promoting the oral availability of nucleoside phosphonate analogs which involves esterification with a lipid to form a lysolecithin mimic. This mimic can utilize natural lysolecithin uptake pathways in the gut, resulting in high oral availability. Since the mimic is not subject to cleavage in the plasma by nonspecific esterases, it remains intact in the circulation and facilitates uptake by target cells. Significant drops in apparent antiviral 50% effective concentrations (EC50s) of up to 3 logs have been observed in comparison with non-lipid-conjugated parent compounds in target cells. We have applied this technology to tenofovir with the goal of increasing oral availability, decreasing the apparent EC50, and decreasing the potential for nephrotoxicity by reducing the exposure of the kidney to the free dianionic tenofovir. Here we report that, in vitro, the hexadecyloxypropyl ester of tenofovir, CMX157, is 267-fold more active than tenofovir against HIV-1 and 4.5-fold more active against HBV. CMX157 is orally available and has no apparent toxicity when given orally to rats for 7 days at doses of 10, 30, or 100 mg/kg/day. Consequently, CMX157 represents a second-generation tenofovir analog which may have an improved clinical profile.

ABSTRACTBiofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility. Staphylococcus epidermidisATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as ≤50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 μg/ml (most >4,096 μg/ml), and four of the experimental compounds had MBICs of ≤128 μg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. epidermidisATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (r= 0.83 to 0.97) and with CFU/ml results (r= 0.85 to 0.94). Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.

ABSTRACTThe antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans.To gain a broader understanding of the cellular response of C. neoformansto auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 μM) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-cajuwas up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

ABSTRACTLinezolid is a member of a new, unique class of synthetic antibacterial agents called oxazolidinones that are effective against gram-positive bacteria, including vancomycin-resistant organisms. We tested the hypothesis that the linezolid clearance would not be altered in subjects with renal dysfunction. Twenty-four subjects with renal function that ranged from normal to severe chronic impairment were enrolled, including patients with end-stage renal disease who were maintained on hemodialysis. Hemodialysis subjects were studied while they were both on and off dialysis. Linezolid was administered as a single oral 600-mg dose, and plasma and urine samples were assayed for linezolid and metabolites for 48 h for all subjects and for up to 96 h for those subjects with impaired renal function not on dialysis. The total apparent oral clearance of linezolid did not change with renal function and ranged from 92.5 to 109.6 ml/min for subjects not requiring dialysis. For subjects on dialysis, the total apparent oral clearance increased from 76.6 ml/min on their off-dialysis day to 130.0 ml/min on their on-dialysis day. Approximately one-third of the dose was removed by dialysis. However, those subjects with severe renal insufficiency (creatinine clearance, <40 ml/min) and those with end-stage renal disease maintained on hemodialysis had higher concentrations of both metabolites. We conclude that no adjustment of the linezolid dosage is needed in subjects with renal dysfunction or subjects on hemodialysis.

ABSTRACTThe mechanism of action of the fungicidal peptide auristatin PHE was investigated in Cryptococcus neoformans. Since auristatin PHE causes budding arrest in C. neoformans(T. Woyke, G. R. Pettit, G. Winkelmann, and R. K. Pettit, Antimicrob. Agents Chemother. 45:3580-3584, 2001), microtubule integrity and nuclear localization in auristatin PHE-treated cells were examined. Iterative deconvolution in conjunction with an optimized C. neoformansmicrotubule immunolabeling procedure enabled detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans. The effect of auristatin PHE on C. neoformansmicrotubule organization was compared with that of the tubulin-binding agent nocodazole. Both drugs produced complete disruption first of cytoplasmic and then of spindle microtubules in a time- and concentration-dependent manner. Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, while 1.5 times the nocodazole MIC was required for the same effect. For both drugs, disruption of microtubules was accompanied by blockage of nuclear migration and of nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage. Nocodazole and the linear peptide auristatin PHE are remarkably different in structure and spectrum of activity, yet on the cellular level, they have similar effects.

ABSTRACTThe pentapeptide dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester (auristatin PHE) is a derivative of the anticancer drug dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine). Broth microdilution assays with a wide variety of yeast and filamentous fungal species demonstrated the specificity of auristatin PHE forCryptococcus neoformansand several species ofTrichosporon. The duration of the postantifungal effect (PAFE) for C. neoformanswas determined for exposure times ranging from 30 min to 2 h. For the derivative, a PAFE was detectable after 45 min of exposure. The effect plateaued after 1 h of exposure, with a PAFE of approximately 6.5 h at four or eight times the auristatin PHE MIC. In contrast, there was no measurable PAFE after 1 h of exposure to dolastatin 10. Human serum greatly prolonged the PAFE of auristatin PHE at eight times the MIC. Auristatin PHE arrested C. neoformansin the budding stage, possibly due to a tubulin-inhibitory action. Auristatin PHE has potential as a narrow-spectrum fungicidal agent and as a probe that can be used to study cryptococcal cell division.

ABSTRACTThe antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with Encephalitozoon intestinalis, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC50s) of pancratistatin and 7-deoxynarciclasine for E. intestinaliswere 0.18 μM and 0.2 μM, respectively, approximately eightfold lower than the IC50s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.

ABSTRACT(−)-β-d-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (−)-β-d-dioxolane guanine (DXG). By using calf adenosine deaminase a Kmvalue of 15 ± 0.7 μM was determined for DAPD, which was similar to theKmvalue for adenosine. However, thekcatfor DAPD was 540-fold slower than thekcatfor adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5′-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2′-deoxyguanosine 5′-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases α and β. Against the large subunit of human DNA polymerase γ aKivalue of 4.3 ± 0.4 μM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.

ABSTRACTSpongistatin 1, a macrocyclic lactone from the marine spongeHyrtios erecta, has broad-spectrum antifungal activity. Since this compound is a potent antimicrotubule agent in mammalian cells, we examined its effects on the filamentous fungusAspergillus nidulansto determine if its antifungal effects are due to antimicrotubule activity. At 25 μg/ml (twice the MIC), spongistatin 1 caused a greater-than-twofold elevation of the chromosome and spindle mitotic indices. Immunofluorescence microscopy revealed that mitotic spindles were smaller and shorter than in control germlings. However, late-anaphase and telophase nuclei were seen occasionally, and this suggests that the spindles are capable of segregating chromosomes. Spongistatin 1 had more dramatic effects on cytoplasmic microtubules. At 30 min after initiation of treatment, 83% of germlings contained fragmented microtubules and after 2 h of treatment, microtubules had disappeared completely from 82% of germlings. In contrast, microtubules disappeared rapidly and completely from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in A. nidulansand that its mechanism of action may involve a novel microtubule-severing activity.

The amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, was evaluated for its acute toxicity in mice and for its efficacy in mice infected with a variety of fungal pathogens. ABLC was markedly less toxic to mice when it was administered intravenously; it had a 50% lethal dose of greater than 40 mg/kg compared with a 50% lethal dose of 3 mg/kg for Fungizone, the desoxycholate form of amphotericin B. ABLC was efficacious against systemic infections in mice caused by Candida albicans, Candida species other than C. albicans, Cryptococcus neoformans, and Histoplasma capsulatum. ABLC was also efficacious in immunocompromised animals infected with C. albicans, Aspergillus fumigatus, and H. capsulatum. Against some infections, the efficacy of ABLC was comparable to that of Fungizone, while against other infections Fungizone was two- to fourfold more effective than ABLC. Against several infections. Fungizone could not be given at therapeutic levels because of intravenous toxicity. ABLC, with its reduced toxicity, could be administered at drug levels capable of giving a therapeutic response. ABLC should be of value in the treatment of severe fungal infections in humans.

ABSTRACTThe biosynthetic peptide dolastatin 10 is currently in phase I and II cancer clinical trials. We evaluated the antifungal spectrum of dolastatin 10 and four structural modifications. In broth macrodilution assays, the peptides were fungicidal for American Type Culture Collection strains and clinical isolates (including fluconazole-resistant strains) of Cryptococcus neoformansbut no other yeasts or filamentous fungi examined. Specificity forC. neoformanswas also demonstrated in the solid-phase disk diffusion assay, and fungicidal activity was confirmed in time-kill experiments. For a methyl ester modification, the MICs at which 50 and 90% of 19 clinical isolates were inhibited (MIC50and MIC90, respectively) were 0.195 and 0.39 μg/ml, respectively. The MFC50(50% minimum fungicidal concentration) for this peptide was 0.39 μg/ml, and the MFC90was 0.78 μg/ml. MICs and MFCs were identical or lower in the presence of human serum but increased with lowered pH. These peptides should be pursued as potential chemotherapeutics forC. neoformans, a leading cause of infection and mortality in immunocompromised patients.

It has recently been reported that penicillin-sensitive pneumococci may exhibit reduced susceptibility to oxacillin, resulting in their misclassification as being penicillin resistant by oxacillin disk testing. Intermediate oxacillin resistance (MIC, 1.0 microgram/ml) in three of these apparently unrelated penicillin-susceptible clinical isolates of Streptococcus pneumoniae isolated in the United Kingdom and in four Spanish isolates was shown to be solely due to the acquisition of a gene encoding an altered penicillin-binding protein (PBP), PBP2X. PBP2X genes cloned from typical penicillin-resistant isolates of S. pneumoniae that possessed high-level oxacillin resistance were shown to be able to transform susceptible isolates of S. pneumoniae to intermediate oxacillin resistance. In all instances, the intermediately oxacillin-resistant PBP2X transformants retained susceptibility to penicillin (MIC, 0.06 microgram/ml). Under appropriate selective pressure, the acquisition of a low-affinity PBP2X by penicillin-susceptible pneumococci could result in an increasing number of false positives for penicillin resistance among isolates of S. pneumoniae screened with oxacillin. Additionally, these intermediately oxacillin-resistant isolates showed reduced susceptibility to cefotaxime, an agent likely to be prescribed in place of penicillin for the treatment of serious infections due to these apparently penicillin-resistant organisms.

Eight clinical isolates of Enterococcus faecium highly resistant to gentamicin (MIC, > 1,000 mg/liter) from patients in six hospitals on three continents were investigated for evidence of spread of either a clone of high-level gentamicin-resistant (HLGR) E. faecium or wide dissemination of a gentamicin resistance (Gmr) plasmid. A combination of ribotypes, plasmid profiles, and extended antimicrobial susceptibilities enabled us to distinguish all but two of the isolates and did not suggest clonal dissemination of a single strain. Two isolates from hospitals situated close together appeared identical by these methods. All of the isolates carried Gmr plasmids which appeared to be closely related following digestion with restriction endonucleases. Cross-hybridization studies confirmed extensive DNA homology between these plasmids. The fragments of these plasmids which hybridized with a probe specific for the aac6'aph2" resistance gene did not resemble those seen in the Gmr transposon Tn5281, which was characterized previously in E. faecalis HH22. This study suggests that there has been widespread dissemination of a single Gmr plasmid and its derivatives amongst isolates of HLGR E. faecium, although a Gmr plasmid from an HLGR E. faecium isolated in the United States showed little homology with the other Gmr plasmids studied.

ABSTRACTWe measured antifungal activity against 128 cryptococcal isolates (86 of C. neoformansand 42 of C. gattii) to determine if differences in serotype susceptibility exist. Contrary to previous results, we found no serotype susceptibility differences. Isavuconazole, posaconazole, and voriconazole demonstrated excellent potency against each isolate and serotype, including isolates with reduced fluconazole susceptibilities.

ABSTRACTWe report a case of Candida glabratainvasive candidiasis that developed reduced susceptibility to caspofungin during prolonged therapy. Pre- and posttreatment isolates were confirmed to be isogenic, and sequencing of hot spots known to confer echinocandin resistance revealed an F659V substitution within the FKS2region of the glucan synthase complex.

ABSTRACTWe compared Etest with broth microdilution testing for isavuconazole activity against 92 Cryptococcusisolates. A 97.8% agreement was found between these methods, without major discrepancies (>2-well dilution difference). Our findings support the use of the Etest methodology as a reliable method for the determination of MICs against Cryptococcusspp.

N4-Hydroxycytidine (NHC) is an antiviral ribonucleoside analog that acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. It exhibits measurable levels of cytotoxicity, with 50% cytotoxic concentration values ranging from 7.5 μM in CEM cells and up to >100 μM in other cell lines.

Patients with coccidioidal meningitis require lifelong antifungal therapy. Cumulative toxicity and lack of antifungal efficacy require salvage therapy in the treatment of some patients.

Fluconazole-induced alopecia is a significant problem for patients receiving long-term therapy. We evaluated the hair cycle changes of fluconazole in a rat model and investigated potential molecular mechanisms.

Carbapenem-resistant Enterobacteriaceae(CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs.

To address the limited bioavailability and intolerance of the conventional itraconazole (ITZ) formulations, a new formulation labeled super bioavailability (SUBA) itraconazole has been developed; however, the specific effects of food and gastric pH are unknown. This study evaluated the pharmacokinetic profile of SUBA itraconazole under fasting and fed conditions, as well as with the concomitant administration of a proton pump inhibitor.

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed.

ABSTRACTThere is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5′-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitrononradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5′-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5′-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5′-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on Dvalues: a safe compound has a Dvalue of >105; a potentially toxic compound has a Dvalue of >104but <105; and a toxic compound has a Dvalue of <104. This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

ABSTRACTWe describe a case of apparent mineralocorticoid excess (AME) secondary to posaconazole therapy and suggest the biochemical mechanism. Clinical and laboratory investigation confirmed 11β-hydroxysteroid dehydrogenase inhibition and withholding therapy led to a resolution of all clinical and laboratory abnormalities. Posaconazole was later restarted at a lower dose and prevented recurrence of this syndrome. Additional studies are necessary to determine the frequency of posaconazole-induced AME and whether other azole antifungals can be associated with this phenomenon.

ABSTRACTZika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitrononradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitroRNA-dependent RNA synthesis in this assay. Our results show that Mn2+is required for enzymatic activity, while Mg2+is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitropolymerase assays in the identification of potential ZIKV inhibitors.

ABSTRACTLarge-scale testing of Coccidioidesisolates has not been performed, and the frequency of clinical isolates with elevated amphotericin B or triazole MICs has not been evaluated. Coccidioidesisolates (n= 581) underwent antifungal susceptibility testing. Elevated MIC values were observed for fluconazole (≥16 μg/ml, 37.3% of isolates; ≥32 μg/ml, 7.9% of isolates), itraconazole (≥2 μg/ml, 1.0% of isolates), posaconazole (≥1 μg/ml, 1.0% of isolates), and voriconazole (≥2 μg/ml, 1.2% of isolates). However, mold-active triazoles exhibited low MICs for the majority of isolates tested. Additional correlation with patient outcomes to determine the relevance of elevated MICs in Coccidioidesisolates is needed.

ABSTRACTThe use of voriconazole, a trifluorinated antifungal, has been associated with the development of fluoride excess and periostitis/exostoses. We evaluated a cohort of patients on long-term triazole therapy and found that other fluorinated triazoles (fluconazole and posaconazole) conferred no risk for the development of hyperfluorosis and its complications in our cohort.

ABSTRACTWe report the attainment of micafungin concentrations from brain tissue and pancreatic pseudocyst fluid from two patients with invasive candidiasis. Micafungin was present in low levels at both body sites, indicating limited penetration into central nervous system (CNS) tissue and pancreatic fluid. Further studies are needed to fully characterize its pharmacokinetics at these locations, as micafungin may potentially serve as an alternative antifungal therapy for CNS or pancreatic candidal infections for which the currently recommended first-line therapy fails.

To determine the role of enterohepatic recirculation on its nonlinear elimination, we studied the enteral absorption of mezlocillin in rabbits. Mezlocillin was not substantially absorbed from the stomach or duodenum. We conclude that enterohepatic recirculation does not cause the dose-dependent elimination kinetics of mezlocillin.