Outcomes by Candida spp. in the ReSTORE Phase 3 trial of rezafungin versus caspofungin for candidemia and/or invasive candidiasis.

Rezafungin is a long-acting, intravenously administered echinocandin for the treatment of candidemia and invasive candidiasis (IC). Non-inferiority of rezafungin vs caspofungin for the treatment of adults with candidemia and/or IC was demonstrated in the Phase 3 ReSTORE study based on the primary endpoints of day 14 global cure and 30-day all-cause mortality. Here, an analysis of ReSTORE data evaluating efficacy outcomes by baseline Candida species is described. Susceptibility testing was performed for Candida species using the Clinical and Laboratory Standards Institute reference broth microdilution method. There were 93 patients in the modified intent-to-treat population who received rezafungin; 94 received caspofungin. Baseline Candida species distribution was similar in the two treatment groups; C. albicans (occurring in 41.9% and 42.6% of patients in the rezafungin and caspofungin groups, respectively), C. glabrata (25.8% and 26.6%), and C. tropicalis (21.5% and 18.1%) were the most common pathogens. Rates of global cure and mycological eradication at day 14 and day 30 all-cause mortality by Candida species were comparable in the rezafungin and caspofungin treatment groups and did not appear to be impacted by minimal inhibitory concentration (MIC) values for either rezafungin or caspofungin. Two patients had baseline isolates with non-susceptible MIC values (both in the rezafungin group: one non-susceptible to rezafungin and one to caspofungin, classified as intermediate); both were candidemia-only patients in whom rezafungin treatment was successful based on the day 30 all-cause mortality endpoint. This analysis of ReSTORE demonstrated the efficacy of rezafungin for candidemia and IC in patients infected with a variety of Candida species.

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.

Multicentre validation of a modified EUCAST MIC testing method and development of associated epidemiologic cut-off (ECOFF) values for rezafungin.

OBJECTIVES: Rezafungin EUCAST MIC testing has been associated with notable inter-laboratory variation, which prevented ECOFF setting for C. albicans. We assessed in vitro susceptibility and reproducibility for a modified EUCAST methodology and established associated wild-type upper limits (WT-ULs). METHODS: MICs against 150 clinical Candida isolates (six species), molecularly characterized fks mutants (n = 13), and QC strains (n = 6) were determined at six laboratories according to E.Def 7.3 but using Tween 20 supplemented medium. WT-ULs were determined using the derivatization method, the ECOFFinder programme and visual inspection. Consensus WT-ULs were determined. RESULTS: The laboratory- and species-specific MIC distributions were Gaussian with >99.5% MICs within four 2-fold dilutions except for C. parapsilosis (92.8%). The following consensus WT-UL were determined: C. albicans 0.008 mg/L; C. dubliniensis and C. glabrata 0.016 mg/L; C. krusei and C. tropicalis 0.03 mg/L; and C. parapsilosis 4 mg/L. Adopting these WT-UL, six clinical isolates were non-wild-type, five of which harboured Fks alterations. For 11/13 mutants, all 670 MICs were categorized as non-wild-type whereas MICs for C. glabrata Fks2 D666Y and C. tropicalis Fks1 R656R/G overlapped with the corresponding wild-type distributions. Repeat testing of six reference strains yielded 98.3%-100% of MICs within three 2-fold dilutions except for C. albicans CNM-CL-F8555 (96%) and C. parapsilosis ATCC 22019 (93.3%). CONCLUSIONS: The modified EUCAST method significantly improved inter-laboratory variation, identified wild-type populations and allowed perfect separation of wild-type and fks mutants except for two isolates harbouring weak mutations. These consensus WT-UL have been accepted as ECOFFs and will be used for rezafungin breakpoint setting.

Evaluation of the Post-Antifungal Effect of Rezafungin and Micafungin against Candida albicans, Candida parapsilosis and Candida glabrata.

Rezafungin, a new echinocandin with an extended half-life, exhibits potent activity against Candida spp. Aside from the MIC, specific interactions between antifungal and isolate, including the duration of anti-infective activity, may impact dose interval choices and infection outcome. We evaluated rezafungin and micafungin post-antifungal effect (PAFE) against C. albicans, C. parapsilosis and C. glabrata. Six Candida spp. isolates were tested, including two of each species, C. albicans, C. parapsilosis and C. glabrata. Antifungal susceptibility testing was performed using the CLSI reference broth microdilution method. Antifungal concentrations of 1x, 4x and 16x the baseline MIC were used for PAFE determinations. Colony counts were performed at T0 (pre-exposure), after the 1-h drug exposure, after the cell wash (T1), and at T2, T4, T8, T12, T24 and T48 h. Rezafungin PAFE results were equivalent to micafungin PAFE values for one C. albicans (>14.9 h) and both C. glabrata (>40 h) isolates for all concentrations tested. The rezafungin and micafungin PAFEs could not be determined against one C. albicans isolate. Prolonged PAFE results were also noted for rezafungin (range, 18.4 to >40 h) against both C. parapsilosis isolates at all concentrations, while no micafungin PAFE or a short PAFE (range, 1.8 to 7.4 h) was observed against these organisms, except at 16x bMIC. Rezafungin showed sustained growth inhibition following drug removal and displayed equivalent or longer PAFE values than micafungin against all tested Candida spp.

In vitro activity of rezafungin against common and rare Candida species and Saccharomyces cerevisiae.

BACKGROUND: Rezafungin is a novel echinocandin with excellent activity against common Candida species; however, limited data are available regarding rare Candida species. METHODS: We determined the in vitro susceptibility of 689 clinical isolates of 5 common and 19 rare Candida species, as well as Saccharomyces cerevisiae. The activity of rezafungin was compared with that of anidulafungin, caspofungin, micafungin, amphotericin B and fluconazole, using CLSI broth microdilution methodology (Fourth Edition: M27). RESULTS: Rezafungin MIC90 values were 0.06 mg/L for Candida albicans (n=125), Candida tropicalis (n=51), Candida dubliniensis (n=22), Candida inconspicua (n=41), Candida sojae (n=10), Candida lipolytica (n=10) and Candida pulcherrima (n=10), 0.12 mg/L for Candida glabrata (n=81), Candida krusei (n=53), Candida kefyr (n=52) and Candida fabianii (n=15), 0.25 mg/L for Candida lusitaniae (n=46) and Candida auris (n=19), 0.5 mg/L for Candida metapsilosis (n=15) and S. cerevisiae (n=21), 1 mg/L for Candida orthopsilosis (n=15) and Candida guilliermondii (n=27) and 2 mg/L for Candida parapsilosis sensu stricto (n=59). Caspofungin MIC90 values were 0.25-2 mg/L for all species, while micafungin and anidulafungin MIC90 values were similar to those of rezafungin. Fluconazole resistance was found in C. albicans (5.6%) and C. glabrata (4.9%); rezafungin was effective against these isolates as well. Amphotericin B MIC values did not exceed 2 mg/L. CONCLUSIONS: Rezafungin showed excellent in vitro activity against both WT and azole-resistant Candida species, as well as against S. cerevisiae. Rezafungin had similar activity to other echinocandins (excluding caspofungin) against common Candida species and, notably, against clinically relevant uncommon Candida species.

Rezafungin (CD101) demonstrates potent in vitro activity against Aspergillus, including azole-resistant Aspergillus fumigatus isolates and cryptic species.

Background: Rezafungin is an investigational echinocandin under development for the treatment and prevention of invasive fungal infections, with a long half-life in humans (∼130 h) and potent in vitro activity against Aspergillus spp. Our objective was to further evaluate its activity against Aspergillus fumigatus isolates, including azole-resistant isolates and cryptic Aspergillus spp. Methods: Clinical isolates of Aspergillus were used, including 15 WT and 31 azole-resistant A. fumigatus, 11 Aspergillus lentulus, 5 each of Aspergillus thermomutatus and Aspergillus udagawae and 11 Aspergillus calidoustus. Minimum effective concentrations (MECs) and MICs of rezafungin, caspofungin, micafungin, posaconazole and voriconazole were determined by CLSI M38-A2 broth microdilution. Differences in geometric mean (GM) MEC/MIC values were assessed for significance by ANOVA. Results: Rezafungin GM MECs for A. fumigatus were 0.024 and 0.043 mg/L for WT and azole-resistant isolates, respectively. Rezafungin was also active against cryptic species, including A. lentulus (0.016 mg/L), A. calidoustus (0.044 mg/L), A. thermomutatus (MEC range ≤0.015-0.25 mg/L) and A. udagawae (≤0.015-0.03 mg/L). This activity was similar to that of caspofungin and micafungin with the exception of A. calidoustus, against which rezafungin was more potent than caspofungin (GM MEC 0.044 versus 0.468 mg/L; P < 0.0001). Conclusions: Rezafungin demonstrated potent in vitro activity against Aspergillus spp., including azole-resistant A. fumigatus isolates and cryptic species with elevated posaconazole and voriconazole MICs. Additional studies are warranted to determine whether the in vitro activity translates into in vivo efficacy against infections caused by resistant Aspergillus isolates.

Time-Kill Kinetics of Rezafungin (CD101) in Vagina-Simulative Medium for Fluconazole-Susceptible and Fluconazole-Resistant Candida albicans and Non-albicans Candida Species.

Background: While echinocandins demonstrate excellent efficacy against Candida species in disseminated infections and demonstrate potent minimal inhibitory concentration (MIC) values under standard susceptibility testing conditions, investigation under conditions relevant to the vaginal environment was needed. We assessed the antifungal activity and time-kill kinetics of the novel echinocandin rezafungin (formerly CD101) under such conditions, against Candida species relevant to vulvovaginal candidiasis (VVC). Methods: Susceptibility testing of fluconazole-susceptible and fluconazole-resistant C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei was performed in RPMI at pH 7.0 and in vagina-simulative medium (VSM) at pH 4.2 for topical rezafungin, terconazole, fluconazole, and amphotericin B. Time-kill kinetics were evaluated for rezafungin and terconazole at 2, 8, 32, and 128 µg/ml over 72 hours. Results: Rezafungin MIC values were the same or 2-fold higher in VSM/pH 4.2 versus RPMI/pH 7.0. Some C. albicans terconazole MIC values were lower, but most were significantly higher in VSM than in RPMI. Rezafungin was fungicidal against 11/14 strains and near-fungicidal against the others. Terconazole (128 µg/ml) was fungicidal against C. krusei and near-fungicidal against susceptible C. parapsilosis but fungistatic versus all other strains evaluated. Conclusion: Rezafungin retained anti-Candida activity and fungicidal activity under in vitro conditions relevant to VVC.

In vitro activity of the novel echinocandin CD101 at pH 7 and 4 against Candida spp. isolates from patients with vulvovaginal candidiasis.

Background: The novel echinocandin CD101 has stability properties amenable to topical formulation for use in the treatment of acute vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC). CD101 has demonstrated potent antifungal activity at pH 7, but assessment of its activity at the physiological pH of the vaginal environment is needed. Objectives: To evaluate the antifungal activity of CD101 against clinical VVC isolates of Candida spp., including azole-resistant strains, at pH 4. Methods: MIC values of CD101 and comparators (fluconazole, itraconazole, micafungin, caspofungin and anidulafungin) were assessed via broth microdilution. MIC assays were conducted at pH 7 and 4 after 24 and 48 h against a 108 VVC isolate panel of Candida spp., including Candida albicans ( n = 60), Candida glabrata ( n = 21), Candida parapsilosis ( n = 14) and Candida tropicalis ( n = 13). Results: Overall, MIC values of all drugs were slightly higher at pH 4 versus 7 and at 48 versus 24 h of incubation. CD101 MIC values typically exhibited ∼4-fold shifts at pH 4 and were not affected by azole susceptibility. C. parapsilosis susceptibility was the least affected at pH 4 and did not increase for most drugs. Conclusions: CD101 had potent activity against all Candida isolates tested, including azole-resistant strains. Although there was some reduction in activity at pH 4 versus 7, the resulting MIC values were still well below the intravaginal CD101 drug concentrations anticipated to be present following topical administration. These results support continued development of topical CD101 for the treatment of VVC/RVVC.

CD101: a novel long-acting echinocandin.

CD101 is a novel echinocandin drug being developed to treat severe fungal infections including invasive candidiasis. We have performed a series of studies to evaluate the antifungal properties of CD101 against both echinocandin-susceptible and -resistant Candida strains. Antifungal susceptibility testing performed on a collection of 95 Candida strains including 30 caspofungin-resistant isolates containing fks mutations demonstrated comparable antifungal potency of CD101 relative to micafungin (MCF) across different Candida species. Comparable kinetic inhibition of glucan synthase activity was also observed for CD101 and MCF on both wild-type (WT) and resistant fks mutant Candida strains. Similarly, both drugs yielded nearly identical values for a mutant prevention concentration. In a murine model of invasive candidiasis, CD101 displayed better or at least comparable efficacy relative to MCF in treating WT or fks mutant Candida albicans. An exceptional long-lived pharmacokinetic profile was observed in mice following a single dose of CD101. Collectively, CD101 has great potential not only in treating invasive Candida infections but also in preventing emergence of resistance to currently approved echinocandin drugs.

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.

Tedizolid for the management of human infections: in vitro characteristics.

The emerging antibiotic resistance of Gram-positive pathogens represents a significant challenge to the management of human infections. The novel oxazolidinone tedizolid demonstrates antimicrobial activity across a broad range of Gram-positive pathogens and greater potency than linezolid against wild-type and drug-resistant pathogens, including linezolid-resistant Staphylococcus aureus strains possessing mutations in chromosomal genes encoding 23S rRNA or ribosomal proteins L3 or L4. Strains harboring such mutations are also selected for much less frequently with tedizolid than with linezolid. In addition, tedizolid has a significant potency advantage over linezolid-resistant strains carrying the horizontally transferable cfr gene. Methylation of A2503 of 23S rRNA by the Cfr methyltransferase confers resistance to linezolid (and a variety of other 50S ribosomal subunit-targeted antibiotics) but not to tedizolid because of structural differences in A-ring C5 substituents between the 2 drugs. The greater potency and improved resistance profile of tedizolid provides the microbiologic basis for considering this molecule as an alternative to linezolid for the treatment of serious 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.

Absorption, distribution, metabolism, and excretion of the novel antibacterial prodrug tedizolid phosphate.

Tedizolid phosphate is a novel antibacterial prodrug with potent activity against Gram-positive pathogens. In vitro and in vivo studies demonstrated that the prodrug is rapidly converted by nonspecific phosphatases to the biologically active moiety tedizolid. Single oral dose radiolabeled (14)C-tedizolid phosphate kinetic studies in human subjects (100 µCi in 204 mg tedizolid phosphate free acid) confirmed a rapid time to maximum tedizolid concentration (Tmax, 1.28 hours), a long terminal half-life (10.6 hours), and a Cmax of 1.99 µg/ml. Metabolite analysis of plasma, fecal, and urine samples from rats, dogs, and humans confirmed that tedizolid is the only measurable metabolite in plasma after intravenous (in animals only) or oral administration and that tedizolid sulfate is the major metabolite excreted from the body. Excellent mass balance recovery was achieved and demonstrated that fecal excretion is the predominant (80-90%) route of elimination across species, primarily as tedizolid sulfate. Urine excretion accounted for the balance of drug elimination but contained a broader range of minor metabolites. Glucuronidation products were not detected. Similar results were observed in rats and dogs after both intravenous and oral administration. The tedizolid metabolites showed less potent antibacterial activity than tedizolid. The observations from these studies support once daily dosing of tedizolid phosphate and highlight important metabolism and excretion features that differentiate tedizolid phosphate from linezolid.

In Vivo Efficacy of Rezafungin, Anidulafungin, Caspofungin, and Micafungin against Four Candida auris Clades in a Neutropenic Mouse Bloodstream Infection Model.

OBJECTIVES: Rezafungin is the first new drug approved to treat candidaemia and invasive candidiasis in more than 10 years. However, data are scant on the in vivo efficacy of rezafungin and the other three approved echinocandins against different Candida auris clades. METHODS: This study involved 10 isolates representing 4 C. auris clades: South Asian (n = 2), East Asian (n = 2), South African (n = 2), and South American (n = 4, including 2 environmental isolates). In the lethality experiment and fungal tissue burden experiment (kidney, heart, and brain), cyclophosphamide-treated BALB/c male mice were intravenously infected (107 and 8 × 106 colony-forming units [CFU]/mouse, respectively). A 20 mg/kg dose of rezafungin was administered on days 1, 3, and 6. Alternatively, beginning 24 h post-infection, mice received 3 mg/kg of caspofungin, 5 mg/kg of micafungin, or 5 mg/kg of anidulafungin once daily for 6 days. RESULTS: Regardless of isolate and clade, all echinocandin regimens improved survival after 21 days (p = 0.0041 to p < 0.0001). All echinocandins frequently produced >3-log mean CFU/g decreases in the fungal kidney and heart burdens, although some of these decreases were not statistically significant. Rezafungin, regardless of clade, produced 3-5 and 2-4 log CFU/g decreases in the kidney and heart burdens, respectively. Echinocandins did not inhibit fungal growth in the brain. Histopathological examination performed on day 7 showed no fungal cells in the heart and kidneys of rezafungin-treated mice and to a lesser extent, caspofungin-treated mice, regardless of the clinical isolate. All echinocandin-treated mice showed medium and/or large foci of fungal cells in their cerebrum or cerebellum. CONCLUSIONS: Regardless of the C. auris clade, rezafungin activity in vivo was comparable to or improved over that of the three previously approved echinocandins.

Anthracimycin, a potent anthrax antibiotic from a marine-derived actinomycete.

Licensed to kill: A new antibiotic, anthracimycin (see scheme), produced by a marine-derived actinomycete in saline culture, shows significant activity toward Bacillus anthracis, the bacterial pathogen responsible for anthrax infections. Chlorination of anthracimycin gives a dichloro derivative that retains activity against Gram-positive bacteria, such as anthrax, but also shows activity against selected Gram-negative bacteria.

Genetic environment and stability of cfr in methicillin-resistant Staphylococcus aureus CM05.

The Cfr methyltransferase confers resistance to many 50S ribosomal subunit-targeted antibiotics, including linezolid (LZD), via methylation of the 23S rRNA base A2503 in the peptidyl transferase center. Methicillin-resistant Staphylococcus aureus strain CM05 is the first clinical isolate documented to carry cfr. While cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon. Here we evaluated the chromosomal locus, association with mobile genetic elements, and stability of the cfr insertion region in CM05. The cfr-containing mlr operon is located within a 15.5-kb plasmid-like insertion into 23S rRNA allele 4. The region surrounding the cfr gene has a high degree of sequence similarity to the broad-host-range toxin/antitoxin multidrug resistance plasmid pSM19035, including a second ermB gene downstream of the mlr locus and istAS-istBS. Analysis of several individual CM05 colonies revealed two distinct populations for which LZD MICs were either 8 or 2 µg/ml. In the LZD(s) colonies (designated CM05Δ), a recombination event involving the two ermB genes had occurred, resulting in the deletion of cfr and the 3' flanking region (cfr-istAS-istBS-ermB). The fitness advantage of CM05Δ over CM05 (though not likely due to the cfr deletion itself) results in the predominance of CM05Δ in the absence of selective pressure. Minicircles resulting from the ermB recombination event and the novel association of cfr with the pSM19035 plasmid system support the potential for the continued dissemination of cfr.

Low fitness cost of the multidrug resistance gene cfr.

The recently described rRNA methyltransferase Cfr that methylates the conserved 23S rRNA residue A2503, located in a functionally critical region of the ribosome, confers resistance to an array of ribosomal antibiotics, including linezolid. A number of reports of linezolid-resistant cfr-positive clinical strains indicate the possible rapid spread of this resistance mechanism. Since the rate of dissemination and the efficiency of maintenance of a resistance gene depend on the fitness cost associated with its acquisition, we investigated the fitness cost of cfr expression in a laboratory Staphylococcus aureus strain. We found that acquisition of the cfr gene does not produce any appreciable reduction in the cell growth rate. Only in a cogrowth competition experiment was some loss of fitness observed because Cfr-expressing cells slowly lose to the cfr-negative control strain. Interestingly, cells expressing wild-type and catalytically inactive Cfr had very similar growth characteristics, indicating that the slight fitness cost associated with cfr acquisition stems from expression of the Cfr polypeptide rather than from the modification of the conserved rRNA residue. In some clinical isolates, cfr is coexpressed with the erm gene, which encodes a methyltransferase targeting another 23S rRNA residue, A2058. Dimethylation of A2058 by Erm notably increases the fitness cost associated with the Cfr-mediated methylation of A2503. The generally low fitness cost of cfr acquisition observed in our experiments with the laboratory S. aureus strain offers a microbiological explanation for the apparent spread of the cfr gene among pathogens.

Comparison of In Vitro Killing Activity of Rezafungin, Anidulafungin, Caspofungin, and Micafungin against Four Candida auris Clades in RPMI-1640 in the Absence and Presence of Human Serum.

Candida auris is an emerging and frequently multidrug-resistant pathogen against which the echinocandins are the preferred therapeutic option. We compared killing activities of anidulafungin, caspofungin, micafungin, and rezafungin against 13 isolates representing four C. auris clades (South Asian n = 3; East Asian n = 3; South African n = 3; South American n = 4, of which two were of environmental origin). Minimum inhibitory concentration MICs and killing kinetics in RPMI-1640 and RPMI-1640 plus 50% serum (50% serum) were determined. The four echinocandins were never fungicidal and induced large aggregates in RPMI-1640 and, less markedly, in 50% serum. Colony forming unit CFU decreases were found more consistently in 50% serum than in RPMI-1640. Isolates from the East Asian clade were killed at ≥1-≥ 4 mg/L with all echinocandins regardless of media. Anidulafungin and micafungin produced killing at peak drug serum concentration (8 mg/L) against environmental but not clinical isolates from the South American and the South African clades. Micafungin at ≥8 mg/L but not anidulafungin produced CFU decreases against the South Asian clade as well. In 50% serum, rezafungin at ≥1-≥ 8 mg/L produced killing against all four clades. The next generation echinocandin, rezafungin, showed the same or better activity at clinically attainable trough concentration regardless of media, compared with anidulafungin, caspofungin, and micafungin against all four tested C. auris clades.

Elevated linezolid resistance in clinical cfr-positive Staphylococcus aureus isolates is associated with co-occurring mutations in ribosomal protein L3.

Resistance to linezolid (LZD) occurs through mutations in 23S rRNA and ribosomal proteins L3 and L4 or through methylation of 23S rRNA by Cfr. Here we report novel L3 mutations, ΔSer145/His146Tyr and ΔMet169-Gly174, co-occurring with cfr in LZD-resistant Staphylococcus aureus isolates recovered from a hospital outbreak in Madrid, Spain. LZD MIC values (16, 32, or 64 µg/ml) correlated with the presence and severity of the L3 mutation. All isolates had TR-700 (torezolid) MIC values of ≤ 2 µg/ml.