Biology subjects
Publications and source records attributed to Chaturvedi, S..
Four pan-resistant Candia auris strains from New York outbreak were 100% inhibited in vitro by combinations of two antifungal drugs using fixed concentrations achievable in vivo. Pan-resistant C. auris strains have mutations in eleven gene targets associated with major antifungal drugs, and constituted a distinct sub-cluster among NY strains.
An ongoing Candida auris outbreak in the New York metropolitan area is the largest recorded to date in North America. Laboratory surveillance revealed NY C. auris isolates are resistant to fluconazole, with variable resistance to other currently used broad-spectrum antifungal drugs, and that several isolates are pan-resistant. Thus, there is an urgent need for new drugs with a novel mechanism of action to combat the resistance challenge. Manogepix (MGX) is a first-in-class agent that targets the fungal Gwt1 enzyme. The prodrug, fosmanogepix, is currently in Phase 2 clinical development for the treatment of fungal infections. We evaluated the susceptibility of 200 New York C. auris isolates to MGX and 10 comparator drugs using CLSI methodology. MGX demonstrated lower MICs than comparators (MIC50 and MIC90 0.03 mg/L; range 0.004-0.06 mg/L). The MGX epidemiological cutoff value (ECV, 99% cutoff) for the tested C. auris isolates was 0.06 mg/L. MGX was 8-32-fold more active than the echinocandins, 16-64-fold more active than the azoles, and 64-fold more active than amphotericin B. No differences were found in the MGX or comparators MIC50, MIC90, or GEOMEAN values when subsets of clinical, surveillance, and environmental isolates were evaluated. The range of MGX MIC values for six C. auris pan-resistant isolates was 0.008-0.015 mg/L, and the median and mode MIC values were 0.015 mg/L, demonstrating that MGX retains activity against these isolates. These data support further clinical evaluation of fosmanogepix for the treatment of C. auris infections, including highly resistant isolates.
Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet-hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Super-resolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein--the major viral transactivator protein--exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency. SIGNIFICANCEProbabilistic bet hedging is a generalized diversification strategy to maximize fitness in unpredictable environments, and has been proposed as an evolutionary basis for herpesvirus latency. However, the molecular mechanisms enabling probabilistic bet hedging have remained elusive. Here, we find that the human herpesvirus cytomegalovirus--a major cause of birth defects and transplant failures--utilizes stochastic variability in the abundance of a protein packaged into individual viral particles to enable probabilistic bet hedging between alternate viral states.
We report low MIC50 of Ibrexafungerp (SCY-078) for 102 Candida auris clinical and surveillance isolates from outbreak in New York. The group included C. auris with a variable resistance to antifungal drugs. Five pan-resistant C. auris isolates were susceptible to Ibrexafungerp with low MIC50 range of 0.12-1 {micro}g/ml.
Genomic outcomes of hybridization depend on selection and recombination in hybrids. Whether these processes have similar effects on hybrid genome composition in contemporary hybrid zones versus ancient hybrid lineages is unknown. Here we show that patterns of introgression in a contemporary hybrid zone in Lycaeides butterflies predict patterns of ancestry in geographically adjacent, older hybrid populations. We find a particularly striking lack of ancestry from one of the hybridizing taxa, Lycaeides melissa, on the Z chromosome in both the old and contemporary hybrids. The same pattern of reduced L. melissa ancestry on the Z chromosome is seen in two other ancient hybrid lineages. More generally, we find that patterns of ancestry in old or ancient hybrids are remarkably predictable from contemporary hybrids, which suggests selection and recombination affect hybrid genomes in a similar way across disparate time scales and during distinct stages of speciation and species breakdown.
Candida auris is a multidrug-resistant yeast which has emerged in healthcare facilities worldwide, however little is known about identification methods, patient colonization, spread, environmental survival, and drug resistance. Colonization on both biotic and abiotic surfaces, along with travel, appear to be the major factors for the spread of this pathogen across the globe. In this investigation, we present laboratory findings from an ongoing C. auris outbreak in NY from August 2016 through 2018. A total of 540 clinical isolates, 11,035 patient surveillance specimens, and 3,672 environmental surveillance samples were analyzed. Laboratory methods included matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for yeast isolate identification, real-time PCR for rapid surveillance sample screening, culture on selective/non-selective media for recovery of C. auris and other yeasts from surveillance samples, antifungal susceptibility testing to determine the C. auris resistance profile, and Sanger sequencing of ribosomal genes for C. auris genotyping. Results included: a) identification and confirmation of C. auris in 413 clinical isolates and 931 patient surveillance isolates, as well as identification of 277 clinical cases and 350 colonized cases from 151 healthcare facilities including 59 hospitals, 92 nursing homes, 1 long-term acute care hospital (LTACH), and 2 hospices, b) successful utilization of an in-house developed C. auris real-time PCR assay for the rapid screening of patient and environmental surveillance samples, c) demonstration of relatively heavier colonization of C. auris in nares compared to the axilla/groin, and d) predominance of the South Asia Clade I with intrinsic resistance to fluconazole and elevated minimum inhibitory concentration (MIC) to voriconazole (81%), amphotericin B (61%), 5-FC (3%) and echinocandins (1%). These findings reflect greater regional prevalence and incidence of C. auris and the deployment of better detection tools in an unprecedented outbreak.
The multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX optimum assay conditions were: DNA extraction at 75{degrees}C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.
Since 2016, New York hospitals and healthcare facilities have faced an unprecedented outbreak of pathogenic yeast Candida auris. We tested over one thousand C. auris isolates from affected facilities and found high-resistance to fluconazole (FLC, MIC50>256 mg/L), and variable resistance to other antifungal drugs. Therefore, we evaluated if two-drug combinations are effective in vitro against multidrug-resistant C. auris. Broth micro-dilution (BMD) plates were custom-designed, and quality controlled by TREK Diagnostic System. We used MIC100 endpoints for the drug combination readings as reported earlier for the intra- and inter-laboratory agreements against Candida species and Aspergillus fumigatus. The study results were derived from 12,960 MIC100 readings, for fifteen C. auris isolates tested against 864 possible two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/L potentiated the most successful combinations with other drugs. Micafungin (MFG), Anidulafungin (AFG), Caspofungin (CAS) at individual concentrations of 0.25 mg/L in combination with 5FC (1.0 mg/L) yielded MIC100 for 14, 13, and 12 of 15 C. auris test isolates. AMB / 5FC (0.25/1.0 mg/L) yielded MIC100 for 13 isolates. None of the combinations were effective for C. auris 18-1, which tested resistant against FLC and 5FC, except POS/5FC (0.12/1.0 mg/L). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to deal with multidrug-resistant C. auris.
Psychrotolerant hyphomycetes including unrecognized taxon are commonly found in bat hibernation sites in Upstate New York. During a mycobiome survey, a new fungal species, Chrysosporium morrisgordonii sp. nov., was isolated from bat White-nose syndrome (WNS) afflicted Graphite mine in Warren County, New York. This taxon was distinguished by its ability to grow at low temperature spectra from 6{degrees}C to 25{degrees}C. Conidia were tuberculate and thick-walled, globose to subglobose, unicellular, 3.5-4.6 {micro}m x3.5-4.6 {micro}m, sessile or borne on narrow stalks. The stalks occurred at right angles to the hypha. Intercalary aleuroconidia were also observed; the hyphae were septate, hyaline, smooth-walled, 1-3 {micro}m wide, straight or branched. The ITS1-5.8S-ITS2 sequence of C. morrisgordonii differed from the closest match to C. speluncarum by 37 nucleotides (94% identity); the Neighbor-joining phylogenetic tree confirmed that C. morrisgordonii was placed in a well-supported monophyletic group with C. speluncarum. LSU and ITS phylogenetic trees further indicated that C. morrisgordonii, C. speluncarum, Renispora flavissima, Neogymnomyces virgineus and Gymnoascus demonbreunii formed a distinct group in the Onygenales.
In less than a decade since its identification in 2009, the emerging fungal pathogen Candida auris has become a major public health threat due to its multidrug resistant (MDR) phenotype, high transmissibility, and high mortality. Unlike any other Candida species, C. auris has acquired high levels of resistance to an already limited arsenal of antifungals. As an emerging pathogen, there are currently a limited number of documented murine models of C. auris infection. These animal models use inoculums as high as 107-108 cells per mouse, and the environmental and occupational exposure of working with these models has not been clearly defined. Using real-time quantitative PCR and culture, we monitored the animal holding room as well as the procedure room for up to six months while working with an intravenous model of C. auris infection. This study determined that shedding of the organism is dose-dependent, as detectable levels of C. auris were detected in the cage bedding when mice were infected with 107 and 108 cells, but not with doses of 105 and 106 cells. Autoclaving bedding in closed micro-isolator cages was found to be an effective way to minimize exposure to animal caretakers. We found that tissue necropsies of infected mice were also an important source of potential source exposure to C. auris. To mitigate these potential exposures, we implemented a rigorous \"buddy system\" workflow and a disinfection protocol that uses 10% bleach followed by 70% ethanol and can be used in any animal facility when using small animal models of C. auris infection.
From microbes to cancers, drug-resistant escape variants cause significant morbidity and mortality1-7. Here we present proof-of-concept that disruption of viral auto-regulatory (feedback) circuits strongly inhibits viral replication and confers an extremely high barrier to the evolution of resistance. Using DNA duplexes, we develop single-molecule feedback-circuit disruptors that interfere with transcriptional negative feedback in human herpesviruses (both Herpes Simplex Virus 1 and Cytomegalovirus) thereby increasing viral transcription factors to cytotoxic levels. Feedback disruptors exhibit low-nanomolar to picomolar IC-50s, reduce viral replication >100-fold in culture and in mice, and synergize with the standard-of-care antivirals. Strikingly, no feedback-disruptor escape mutants evolved over >60 days of culture, in contrast to approved antivirals to which resistance rapidly evolved. Overall, the results demonstrate that molecular targeting of feedback circuitry could yield escape-resistant antivirals, potentially enabling development of a new class of antimicrobials.
A fundamental signal-processing problem is how biological systems maintain phenotypic states (i.e., canalization) long after degradation of initial catalyst signals. For example, to efficiently replicate, herpesviruses (e.g., human cytomegalovirus, HCMV) rapidly counteract cell-mediated silencing using trans-activators packaged in the tegument of the infecting virion particle. But, the activity of these tegument trans-activators is inherently transient--they undergo immediate proteolysis but delayed synthesis--and how transient activation sustains lytic viral gene expression despite cell-mediated silencing is unclear. By constructing a two-color, conditional-feedback HCMV mutant, we find that positive feedback in HCMVs Immediate Early 1 (IE1) protein is of sufficient strength to sustain HCMV lytic expression. Single-cell time-lapse imaging and mathematical modeling show that IE1 positive feedback converts transient transactivation signals from tegument pp71 proteins into sustained lytic expression, which is obligate for efficient viral replication, whereas attenuating feedback decreases fitness by promoting a reversible silenced state. Together, these results identify a regulatory mechanism enabling herpesviruses to sustain expression despite transient activation signals--akin to early electronic transistors--and expose a potential target for therapeutic intervention.
Despite accumulating evidence that evolution can be predictable, studies quantifying the predictability of evolution remain rare. Here, we measured the predictability of genome-wide evolutionary changes associated with a recent host shift in the Melissa blue butterfly (Lycaeides melissa). We asked whether and to what extent genome-wide patterns of evolutionary change in nature could be predicted (1) by comparisons among instances of repeated evolution, and (2) from SNP x performance associations in a lab experiment. We delineated the genetic loci (SNPs) most strongly associated with host use in two L. melissa lineages that colonized alfalfa. Whereas most SNPs were strongly associated with host use in none or one of these lineages, we detected a ~two-fold excess of SNPs associated with host use in both lineages. Similarly, we found that host-associated SNPs in nature could also be partially predicted from SNP x performance (survival and weight) associations in a lab rearing experiment. But the extent of overlap, and thus degree of predictability, was somewhat reduced. Although we were able to predict (to a modest extent) the SNPs most strongly associated with host use in nature (in terms of parallelism and from the experiment), we had little to no ability to predict the direction of evolutionary change during the colonization of alfalfa. Our results show that different aspects of evolution associated with recent adaptation can be more or less predictable, and highlight how stochastic and deterministic processes interact to drive patterns of genome-wide evolutionary change.