Development and preliminary validation of a modified EUCAST yeast broth microdilution MIC method with Tween 20-supplemented medium for rezafungin.

BACKGROUND: Rezafungin is a novel, once-weekly echinocandin. EUCAST rezafungin MIC testing has been associated with a good separation of WT and target gene mutant isolates in single-centre studies, but an unacceptable inter-laboratory MIC variation has prevented EUCAST breakpoint setting. This has been attributed to non-specific binding to surfaces across microtitre plates, pipettes, reservoirs, etc. used, as previously encountered for some antibiotics. OBJECTIVES: To investigate use of a surfactant to mitigate non-specific binding of rezafungin in EUCAST E.Def 7.3 MIC testing. METHODS: Surfactants including Tween 20 (T20), Tween 80 (T80) and Triton X-100 (TX100) were evaluated for stand-alone or synergistic antifungal activity via checkerboard assays in combination with rezafungin. Subsequent T20 studies defined an optimized assay concentration, validated in up to four microtitre plate types for WT and fks mutant Candida strains (seven species total) and the six-strain EUCAST Candida quality control (QC) panel. Lastly, T20 inter-manufacturer variability, thermostability and best handling practices were investigated. RESULTS: T20 and T80 performed equivalently, with characteristics slightly preferable to TX100. Due to existing use in EUCAST mould susceptibility testing, T20 was pursued. An optimized concentration of 0.002% T20 normalized rezafungin MIC values across plate types for all Candida spp. evaluated, maintained differentiation of WT versus fks mutants and generated robust QC ranges. Additionally, T20 performance was consistent across manufacturers and temperatures. T20 can be reliably transferred utilizing a syringe, wide-orifice pipette tip and/or by mass. CONCLUSIONS: Supplementation of RPMI (Roswell Park Memorial Institute) 1640 medium with 0.002% T20 generated a highly reproducible EUCAST yeast MIC methodology for rezafungin.

20S Proteasome as a Drug Target in Trichomonas vaginalis.

Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven ß subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the ß1 and ß5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.

Composite thermoresponsive hydrogel with auranofin-loaded nanoparticles for topical treatment of vaginal trichomonad infection.

Trichomonas vaginalis is responsible for the most common non-viral sexually-transmitted disease worldwide. Standard treatment is with oral nitro-heterocyclic compounds, metronidazole or tinidazole, but resistance to these drugs is emerging and adverse effects can be problematic. Topical treatment offers potential benefits for increasing local drug concentrations and efficacy, while reducing systemic drug exposure, but no topical strategies are currently approved for trichomoniasis. The anti-rheumatic drug, auranofin (AF), was recently discovered to have significant trichomonacidal activity, but has a long plasma half-life and significant adverse effects. Here, we used this drug as a model to develop a novel topical formulation composed of AF-loaded nanoparticles (NP) embedded in a thermoresponsive hydrogel for intravaginal administration. The AF-NP composite gel showed sustained drug release for at least 12 h, and underwent sol-gel transition with increased viscoelasticity within a minute. Intravaginal administration in mice showed excellent NP retention for >6 h and markedly increased local AF levels, but reduced plasma and liver levels compared to oral treatment with a much higher dose. Furthermore, intravaginal AF-NP gel greatly outperformed oral AF in eliminating vaginal trichomonad infection in mice, while causing no systemic or local toxicity. These results show the potential of the AF-NP hydrogel formulation for effective topical therapy of vaginal infections.

Characterization of In Vitro Resistance Development to the Novel Echinocandin CD101 in Candida Species.

CD101 is a novel echinocandin with a long half-life undergoing clinical development for treatment of candidemia/invasive candidiasis and vulvovaginal candidiasis. The potential for and mechanisms underlying the development of resistance to CD101 in Candida species were investigated by using spontaneous resistance and serial passage selection methodologies. Four Candida spp. (C. albicans, C. glabrata, C. parapsilosis, and C. krusei) were chosen for resistance characterization with CD101, anidulafungin, and caspofungin. The frequency of spontaneous, single-step mutations conferring reduced susceptibility to CD101 at 1× the agar growth inhibition concentration was low across all species, with median frequencies ranging from 1.35 × 10(-8) to 3.86 × 10(-9), similar to ranges generated for anidulafungin and caspofungin. Serial passage of Candida spp. on agar plates containing drug gradients demonstrated a low potential for resistance development, with passage 20 CD101-selected strains possessing increases in MICs equivalent to or lower than those for the majority of strains generated under selection with anidulafungin and caspofungin. A total of 12 fks "hot spot" mutations were identified, typically in strains with the highest MIC shifts. Cross-resistance was broadly observed among the 3 echinocandins evaluated, with no CD101-selected mutants (with or without fks hot spot mutations) exhibiting reduced susceptibility to CD101 but not also to anidulafungin and/or caspofungin. Consistent with currently approved echinocandins, CD101 demonstrates a low potential for resistance development, which could be further enhanced in vivo by the high maximum concentration of drug in serum (Cmax)/area under the concentration-time curve (AUC) plasma drug exposure achieved with once-weekly dosing of CD101.

Results of the surveillance of Tedizolid activity and resistance program: in vitro susceptibility of gram-positive pathogens collected in 2011 and 2012 from the United States and Europe.

The in vitro activity and spectrum of tedizolid and comparators were analyzed against 6884 Gram-positive clinical isolates collected from multiple US and European sites as part of the Surveillance of Tedizolid Activity and Resistance Program in 2011 and 2012. Organisms included 4499 Staphylococcus aureus, 537 coagulase-negative staphylococci (CoNS), 873 enterococci, and 975 ß-hemolytic streptococci. The MIC values that inhibited 90% of the isolates within each group (MIC90) were 0.25 µg/mL for Staphylococcus epidermidis and ß-hemolytic streptococci and 0.5 µg/mL for S. aureus, other CoNS, and enterococci. Of 16 isolates with elevated tedizolid or linezolid MIC values (intermediate or resistant isolates), 10 had mutations in the genes encoding 23S rRNA (primarily G2576T), 5 had mutations in the genes encoding ribosomal proteins L3 or L4, and 5 carried the cfr multidrug resistance gene. Overall, tedizolid showed excellent activity against Gram-positive bacteria and was at least 4-fold more potent than linezolid against wild-type and linezolid-resistant isolates. Given the low overall frequency of isolates that would be resistant to tedizolid at the proposed break point of 0.5 µg/mL (0.19%) and potent activity against contemporary US and European isolates, tedizolid has the potential to serve as a valuable therapeutic option in the treatment of infections caused by Gram-positive pathogens.

Linezolid-resistant Staphylococcus aureus strain 1128105, the first known clinical isolate possessing the cfr multidrug resistance gene.

The Cfr methyltransferase confers resistance to six classes of drugs which target the peptidyl transferase center of the 50S ribosomal subunit, including some oxazolidinones, such as linezolid (LZD). The mobile cfr gene was identified in European veterinary isolates from the late 1990s, although the earliest report of a clinical cfr-positive strain was the 2005 Colombian methicillin-resistant Staphylococcus aureus (MRSA) isolate CM05. Here, through retrospective analysis of LZD(r) clinical strains from a U.S. surveillance program, we identified a cfr-positive MRSA isolate, 1128105, from January 2005, predating CM05 by 5 months. Molecular typing of 1128105 revealed a unique pulsed-field gel electrophoresis (PFGE) profile most similar to that of USA100, spa type t002, and multilocus sequence type 5 (ST5). In addition to cfr, LZD resistance in 1128105 is partially attributed to the presence of a single copy of the 23S rRNA gene mutation T2500A. Transformation of the ∼37-kb conjugative p1128105 cfr-bearing plasmid from 1128105 into S. aureus ATCC 29213 background strains was successful in recapitulating the Cfr antibiogram, as well as resistance to aminoglycosides and trimethoprim. A 7-kb cfr-containing region of p1128105 possessed sequence nearly identical to that found in the Chinese veterinary Proteus vulgaris isolate PV-01 and in U.S. clinical S. aureus isolate 1900, although the presence of IS431-like sequences is unique to p1128105. The cfr gene environment in this early clinical cfr-positive isolate has now been identified in Gram-positive and Gram-negative strains of clinical and veterinary origin and has been associated with multiple mobile elements, highlighting the versatility of this multidrug resistance gene and its potential for further dissemination.

Identification and characterization of linezolid-resistant cfr-positive Staphylococcus aureus USA300 isolates from a New York City medical center.

The cfr gene was identified in three linezolid-resistant USA300 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected over a 3-day period at a New York City medical center in 2011 as part of a routine surveillance program. Each isolate possessed a plasmid containing a pSCFS3-like cfr gene environment. Transformation of the cfr-bearing plasmids into the S. aureus ATCC 29213 background recapitulated the expected Cfr antibiogram, including resistance to linezolid, tiamulin, clindamycin, and florfenicol and susceptibility to tedizolid.

Broad dissemination of plasmid-mediated quinolone resistance genes in sediments of two urban coastal wetlands.

Contamination of soil and water with antibiotic-resistant bacteria may create reservoirs of antibiotic resistance genes that have the potential to negatively impact future public health through horizontal gene transfer. The plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrS, qepA, and aac(6')-Ib-cr were detected by PCR amplification of metagenomic DNA from surface sediments of the Tijuana River Estuary, a sewage-impacted coastal wetland along the U.S.-Mexico border; sediments of Famosa Slough, a nearby urban wetland that is largely unaffected by sewage, contained only qnrB, qnrS, and qepA. The number of PCR-positive sites and replicates increased in both wetlands after rainfall. Real-time quantitative PCR revealed a significant increase (p < 0.0005) in qnrA abundance (copies per gram sediment or per 16S rDNA copy) in Tijuana River Estuary sediments immediately following rainfall, but no significant change was measured at Famosa Slough (p > 0.1). Nucleotide sequences of cloned qnrA amplicons were all affiliated with qnrA genes found on plasmids of clinical isolates with one exception that was most similar to the chromosomal qnrA gene found in Shewanella algae. Our results suggest that urban wetlands may become reservoirs of antibiotic resistance genes, particularly where wastewater is improperly managed.