scholarly journals Forward and reverse genetic dissection of morphogenesis identifies filament-competent Candida auris strains

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Darian J. Santana ◽  
Teresa R. O’Meara
Keyword(s):  
Transformation Efficiency ◽  
Antifungal Susceptibility ◽  
Genetic Regulation ◽  
Genetic Dissection ◽  
Reverse Genetic ◽  
Growth Morphology ◽  
Candida Auris ◽  
Global Concern ◽  
Healthcare Associated ◽  
Cellular Aggregates

AbstractCandida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome.

scholarly journals Forward and Reverse Genetic Dissection of Morphogenesis Identifies Filament-Competent Candida auris Strains

2021 ◽  
Author(s):  
Darian J. Santana ◽  
Teresa R. O’Meara
Keyword(s):  
Homologous Recombination ◽  
Fungal Pathogen ◽  
Genetic Manipulation ◽  
Expression System ◽  
Genetic Regulation ◽  
Morphological Plasticity ◽  
Genetic Dissection ◽  
Reverse Genetic ◽  
Candida Auris ◽  
Targeted Integration

AbstractCandida auris is an emerging healthcare-associated pathogen of global concern. Although this organism does not display the same morphological plasticity as the related fungal pathogen Candida albicans, recent reports have identified numerous C. auris isolates that grow in cellular aggregates or filaments. However, the genetic circuitry governing C. auris morphology remains largely uncharacterized. Here, we developed an Agrobacterium-mediated transformation system to generate mutants exhibiting aggregating or filamentous cell morphologies. Aggregating strains were associated with disruption of homologs of Saccharomyces cerevisiae chitinase and chitin synthase regulatory proteins, including components of the Regulation of ACE2 Morphogenesis (RAM) pathway, while disruption of a homolog of the S. cerevisiae ELM1 gene resulted in a novel filamentous strain of C. auris. To facilitate targeted genetic manipulation, we developed a transiently expressed Cas9 and sgRNA expression system for use in C. auris. Transformation using this system significantly increased the efficiency of homologous recombination and targeted integration of a reporter cassette in all four clades of C. auris. Using this system, we generated targeted deletion mutants to confirm the roles of RAM and Elm1 proteins in regulating C. auris morphogenesis. Overall, our findings provide novel insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic manipulation tools described here will allow for inexpensive and efficient manipulation of the C. auris genome.ImportanceCandida auris is an emerging and often multi-drug resistant fungal pathogen responsible for outbreaks globally. Current difficulties in performing genetic manipulation in this organism remain a barrier to understanding C. auris biology. Homologous recombination approaches can result in less than 1% targeted integration of a reporter cassette, emphasizing the need for new genetic tools specific for manipulating C. auris. Here, we adapted Agrobacterium-mediated transformation and a transient Cas9 and sgRNA expression system for use in forward and reverse genetic manipulation of C. auris. We demonstrated the efficacy of each system by uncovering genes underlying cellular morphogenesis in C. auris. We identified a novel filamentous mutant of C. auris, demonstrating that this organism has maintained the capacity for filamentous growth. Our findings provide additional options for improving the genetic tractability of C. auris, which will allow for further characterization of this emerging pathogen.

scholarly journals Shifting Landscape of Healthcare-Associated Infection and Antimicrobial Resistant Infection Reporting Policy, 2005–2019

2020 ◽  
Vol 41 (S1) ◽  
pp. s389-s389
Author(s):  
Jeremy Goodman ◽  
Samuel Clasp ◽  
Arjun Srinivasan ◽  
Elizabeth Mothershed ◽  
Seth Kroop ◽  
...  
Keyword(s):  
Health Department ◽  
Adverse Outcomes ◽  
Health Departments ◽  
Healthcare Associated Infection ◽  
Candida Auris ◽  
Quality Reporting ◽  
Notifiable Diseases ◽  
Reporting Requirements ◽  
Resistant Infection ◽  
Healthcare Associated

Background: Healthcare-associated infections (HAIs) are a serious threat to patient safety; they account for substantial morbidity, mortality, and healthcare costs. Healthcare practices, such as inappropriate use of antimicrobials, can also amplify the problem of antimicrobial resistance. Data collected to target HAI prevention and antimicrobial stewardship efforts and measure progress are an important resource for assuring transparency and accountability in healthcare, tracking adverse outcomes, investigating healthcare practices that may spread or protect against disease, detecting and responding to the spread of resistant pathogens, preventing infections, and saving lives. Methods: We discuss 3 healthcare-associated infection and antimicrobial Resistant infection (HAI-AR) reporting types: NHSN HAI-AR reporting, reportable diseases, and nationally notifiable diseases. HAI-AR reporting requirements outline facilities and data to report to NHSN and the health department to comply with state laws. Reportable diseases are those that facilities, providers, and laboratories are required to report to the health department. Nationally notifiable diseases are those reported by health departments to the CDC for nationwide surveillance and analysis as determined by Council of State and Territorial Epidemiologists (CSTE) and the CDC. Data presented are based on state and federal policy; NHSN data are based on CDC reporting statistics. Results: Since the 2005 launch of the CDC NHSN and publication of federal advisory committee HAI reporting guidance, most states have established policies stipulating healthcare facilities in their jurisdiction report HAIs and resistant infections to the NHSN to gain access to those data, increasing from 2 states in 2005, to 18 in 2010, and to 36 states, Washington, DC, and Philadelphia in 2019. Reporting policies and NHSN participation expanded greatly following the 2011 inception of CMS HAI quality reporting requirements, with several states aligning state requirements with CMS reporting. States listing carbapenem-resistant Enterobacteriaceae (CRE) as a reportable disease increased from 7 in 2013 to 41 states and the District of Columbia in 2019. Vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus (VISA/VRSA) was added as a nationally notifiable disease in 2004, carbapenemase-producing CRE (CP-CRE) was added in 2018, and Candida auris clinical infections were added in 2019. The CDC and most jurisdictions with HAI reporting mandates issue public reports based on aggregate state data and/or facility-level data. States may also alert healthcare providers and health departments of emerging threats and to assist in notifying patients of potential exposure. Conclusions: Through efforts by health departments, facilities, patient advocates, partners, the CDC, and other federal agencies, HAI-AR reporting has steadily increased. Although reporting laws and data uses vary between jurisdictions, data provided serves as valuable tools to inform prevention.Funding: NoneDisclosures: None

scholarly journals Two Candida auris Cases in Germany with No Recent Contact to Foreign Healthcare—Epidemiological and Microbiological Investigations

2021 ◽  
Vol 7 (5) ◽  
pp. 380
Author(s):  
Joerg Steinmann ◽  
Thomas Schrauzer ◽  
Lisa Kirchhoff ◽  
Jacques F. Meis ◽  
Peter-Michael Rath
Keyword(s):  
South Asian ◽  
Multidrug Resistant ◽  
Health Care Facilities ◽  
Candida Auris ◽  
Contact Precautions ◽  
Asian Clade ◽  
Care Facilities ◽  
Long Time ◽  
European Health ◽  
Healthcare Associated

Candida auris has become a global fungal public health threat. This multidrug-resistant yeast is associated with nosocomial intra- and interhospital transmissions causing healthcare-associated infections. Here, we report on two C. auris cases from Germany. The two patients stayed in Germany for a long time before C. auris was detected during their hospitalization. The patients were isolated in single rooms with contact precautions. No nosocomial transmissions were detected within the hospital. Both C. auris isolates exhibited high minimum inhibitory concentrations (MICs) of fluconazole and one isolate additionally high MICs against the echinocandins. Microsatellite genotyping showed that both strains belong to the South Asian clade. These two cases are examples for appropriate in-hospital care and infection control without further nosocomial spread. Awareness for this emerging, multidrug-resistant pathogen is justified and systematic surveillance in European health care facilities should be performed.

scholarly journals Emergence of Candida auris in Brazil in a COVID-19 Intensive Care Unit

2021 ◽  
Vol 7 (3) ◽  
pp. 220
Author(s):  
João N. de Almeida ◽  
Elaine C. Francisco ◽  
Ferry Hagen ◽  
Igor Brandão ◽  
Felicidade M. Pereira ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Antifungal Susceptibility ◽  
Candida Auris ◽  
Minimal Inhibitory Concentrations ◽  
First Case ◽  
Asian Clade ◽  
Travel Restrictions ◽  
Clade C ◽  
The City

In December 2020, Candida auris emerged in Brazil in the city of Salvador. The first two C. auris colonized patients were in the same COVID-19 intensive care unit. Antifungal susceptibility testing showed low minimal inhibitory concentrations of 1 µg/mL, 2 µg/mL, 0.03 µg/L, and 0.06 µg/mL for amphotericin B, fluconazole, voriconazole, and anidulafungin, respectively. Microsatellite typing revealed that the strains are clonal and belong to the South Asian clade C. auris. The travel restrictions during the COVID-19 pandemic and the absence of travel history among the colonized patients lead to the hypothesis that this species was introduced several months before the recognition of the first case and/or emerged locally in the coastline Salvador area.

scholarly journals Admission Screening for Candida auris Among High-Risk Patient Populations

2020 ◽  
Vol 41 (S1) ◽  
pp. s112-s113
Author(s):  
Christine D. Spalding ◽  
Zelazny Adrian ◽  
Christina M. Kenosky ◽  
Shamira J. Shallom ◽  
Seyedmojtaba Syedmoussavi ◽  
...  
Keyword(s):  
High Risk ◽  
High Risk Patient ◽  
The United States ◽  
Risk Patient ◽  
Molecular Testing ◽  
Candida Auris ◽  
Admission Screening ◽  
Clinical Center ◽  
Its Sequence Analysis ◽  
Healthcare Associated

Background:Candida auris is a highly transmissible healthcare-associated pathogen that can cause severe infection as well as long-lasting colonization. C. auris is often resistant to the antifungals that are commonly used to treat Candida infections, which may lead to clinical failure. Therefore, healthcare facilities must identify the organism quickly and implement strict precautions to prevent its spread. In 2019, the NIH Clinical Center instituted C. auris admission screening among its high-risk patient populations. Methods: Patients admitted to the NIH Clinical Center, a 200-bed research hospital, were identified on admission as having been hospitalized outside the United States in the prior 6 months. Admission screening began in August 2019. In September 2019, due to evolving regional epidemiology, we expanded surveillance criteria to include patients housed in any healthcare facility in the District of Columbia, Maryland, and Virginia metro area in the previous 6 months. Screening was performed as routine clinical care, and therefore did not require written informed consent. Swabs were obtained from nares, axilla and groin, with subsequent addition of mouth and toe web (BD ESwabs). Patients were placed on empiric contact isolation for at least 48 hours and concurrently screened for carbapenemase-producing organisms. Swabs were cultured on CHROMagar Candida and in Sabouraud dextrose broth with 10% NaCL and 50 mg/L chloramphenicol and gentamicin, and incubated for 14 days at 30°C and 40°C, respectively. Positive broth tubes were subcultured onto CHROMagar Candida. C. auris was identified by MALDI-TOF MS and ITS sequence analysis. Susceptibility testing was performed using Sensititre YeastOne Colorimetric assay. Whole-genome sequencing was used to identify clonal designations and genetic relatedness of isolates. Results: Since August 2019, 1 to 2 patients per week have been screened for C. auris. As of November 2019, 1 of 15 patients screened on admission grew C. auris from a groin swab. The patient, who had been hospitalized abroad, was found to be cocolonized with blaNDM-1+ E. coli and K. pneumoniae. Subsequent screening of other patients on the same ward identified no evidence of spread. Admission surveillance is ongoing. Conclusions: Healthcare-associated outbreaks can originate from C. auris–colonized patients. Admission surveillance of high-risk patients is intended to prevent transmission from undetected reservoirs. Our sampling of multiple sites, though laborious, may add to the data on C. auris colonization. Future plans include incorporating molecular testing and streamlining geographic criteria. C. auris admission screening has already identified one colonized patient, and will continue as a new and important patient safety measure at our hospital.Funding: NoneDisclosures: None

scholarly journals CYTOGENETIC ANALYSIS OF THE CHROMOSOMAL REGION IMMEDIATELY ADJACENT TO THE ROSY LOCUS IN DROSOPHILA MELANOGASTER

Genetics ◽  
1980 ◽  
Vol 95 (1) ◽  
pp. 95-110 ◽  
Author(s):  
Arthur J Hilliker ◽  
Stephen H Clark ◽  
Arthur Chovnick ◽  
William M Gelbart
Keyword(s):  
Drosophila Melanogaster ◽  
Polytene Chromosome ◽  
Structural Information ◽  
Genetic Regulation ◽  
Chromosome Band ◽  
Genetic Dissection ◽  
Genetic Unit ◽  
Complementation Groups ◽  
The Relationship ◽  
Chromosome Bands

ABSTRACT This report describes the genetic analysis of a region of the third chromosome of Drosophila melanogaster extending from 87D2-4 to 87E12-F1, an interval of 23 or 24 polytene chromosome bands. This region includes the rosy (ry, 3-52.0) locus, carrying the structural information for xanthine dehydrogenase (XDH). We have, in recent years, focused attention on the genetic regulation of the rosy locus and, therefore, wished to ascertain in detail the immediate genetic environmcnt of this locus. Specifically, we question if rosy is a solitary genetic unit or part of a larger complex genetic unit encompassing adjacent genes. Our data also provide opportunity to examine further the relationship between euchromatic gene distrihution and polytene chromosome structure.—The results of our genetic dissection of the rosy microregion substantiate the conclusion drawn earlier (SCHALET, KERNAGHAN and CHOVNICK 1964) that the rosy locus is the only gene in this region concerned with XDH activity and that all adjacent genetic units are functionally, as well as spatially, distinct Erom the rosy gene. Within the rosy micro-region, we observed a close correspondence between the number of complementation groups (21) and the number of polytene chromosome bands (23 or 24). Consideration of this latter observation in conjunction with those of similar studies of other chhromosomal regions supports the hypothesis that each polytene chromosome band corresponds to a single genetic unit.

scholarly journals Candida auris Clinical Isolates from South Korea: Identification, Antifungal Susceptibility, and Genotyping

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Yong Jun Kwon ◽  
Jong Hee Shin ◽  
Seung A Byun ◽  
Min Ji Choi ◽  
Eun Jeong Won ◽  
...  
Keyword(s):  
Antifungal Susceptibility ◽  
Diagnostic Tools ◽  
Candida Auris ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Fluconazole Resistant ◽  
Vitek 2 ◽  
Vitek Ms ◽  
Tof Ms

ABSTRACT Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.

scholarly journals Farnesol abrogates biofilm- and efflux pump- associated genes in drug resistant Candida auris strains

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Vartika Srivastava ◽  
Aijaz Ahmad
Keyword(s):  
Active Drug ◽  
Efflux Pump ◽  
Antifungal Susceptibility ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Major Facilitator Superfamily ◽  
Drug Efflux ◽  
Candida Auris ◽  
Efflux Mechanism ◽  
Major Facilitator

Background: Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and over expression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. Methods: In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. Rhodamine-6-G extracellular efflux and intracellular accumulation assays were used to study active drug efflux mechanism. We further studied the role of farnesol in modulating development of biofilms and drug efflux in C. auris. Down-regulation of biofilm- and efflux pump- associated genes by farnesol was also investigated. CLSM analysis for examining C. auris biofilm architecture among treated and untreated isolates. Results: Most of the isolates (twenty-two) were found resistant to FLZ whereas five were resistant to AmB. All the isolates were found capable of biofilm formation and ornamented with active drug efflux mechanism. The MIC for planktonic cells ranged from 62.5-125 mM and for sessile cells was 125 mM (0 h and 4 h biofilm) and 500 mM (12 h and 24 h biofilm), CLSM studies also confirmed these findings. Farnesol also blocked efflux pumps and down-regulated biofilm- and efflux pump- associated genes. Conclusion: Modulation of biofilm- and efflux pump- associated genes by farnesol represent a promising approach in combating C. auris infection.

scholarly journals 680. Month Long Fungemia due to Candida auris Endocarditis

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S443-S443
Author(s):  
Haseeba khan ◽  
Christy Varughese ◽  
Hemil Gonzalez
Keyword(s):  
Fungal Infections ◽  
Antifungal Susceptibility ◽  
Acute Care Hospital ◽  
Sigmoid Resection ◽  
Blood Cultures ◽  
Multi Drug Resistance ◽  
Care Hospital ◽  
Candida Auris ◽  
Candida Spp ◽  
First Case

Abstract Background Candida auris (C. auris) is a multidrug resistant Candida species, reported to cause persistent fungemia along with a multitude of invasive fungal infections. We report the first case of C. auris fungemia due to endocarditis. Methods 61 year old man with a history of diverticulitis that required sigmoid resection and was complicated by abdominal abscesses due to multi drug resistant organisms warranting heavy antibiosis. Prolonged hospitalisation for that surgery was followed by a stay at a long term acute care hospital. He was readmitted at an outside hospital with sepsis where blood cultures grew C.auris. Upon evaluation, was found to have aortic valve endocarditis. Per patient’s preference, surgery was initially deferred. Despite escalation of therapy with a combination of antifungals, he remained fungemic for five weeks with repeat blood cultures showing changing antifungal susceptibility patterns. Patient eventually underwent surgical intervention at our facility, with valve cultures being positive for C.auris. After the surgery he was treated with 6 weeks of intravenous combination antifungal therapy. Results C.auris’s pathogenicity stems from multiple mechanisms with multi drug resistance being most pertinent. What adds to the complexity of the management is the absence of C.auris specific minimum inhibitory concentration breakpoints. Therefore treatment is based on Center for Disease Control’s (CDC) proposed breakpoints that have been extrapolated from other Candida spp. It is further complicated by lack of C.auris specific data showing essential agreement among different commercially available antifungal susceptibility testing (AFST). Heteroresistance of the microbial population is an issue that must be considered in such protracted fungemia. Conclusion Invasive infections due to Candida auris presents as a diagnostic and therapeutic challenge to clinicians. Disclosures All Authors: No reported disclosures

scholarly journals Echinocandins as Biotechnological Tools for Treating Candida auris Infections

2020 ◽  
Vol 6 (3) ◽  
pp. 185
Author(s):  
Elizabete de Souza Cândido ◽  
Flávia Affonseca ◽  
Marlon Henrique Cardoso ◽  
Octavio Luiz Franco
Keyword(s):  
Fungal Pathogen ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Healthcare Associated Infections ◽  
Candida Auris ◽  
High Interest ◽  
The Past ◽  
Recent Emergence ◽  
Echinocandin Resistance ◽  
Healthcare Associated

Candida auris has been reported in the past few years as an invasive fungal pathogen of high interest. Its recent emergence in healthcare-associated infections triggered the efforts of researchers worldwide, seeking additional alternatives to the use of traditional antifungals such as azoles. Lipopeptides, specially the echinocandins, have been reported as an effective approach to control pathogenic fungi. However, despite its efficiency against C. auris, some isolates presented echinocandin resistance. Thus, therapies focused on echinocandins’ synergism with other antifungal drugs were widely explored, representing a novel possibility for the treatment of C. auris infections.

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