Drug Resistance In Yeast Research Articles

In vitro and silico activity of piperlongumine against azole-susceptible/resistant Aspergillus fumigatus and terbinafine-susceptible/resistant Trichophyton species

In recent years, the widespread emergence of drug resistance in yeasts and filamentous fungi to existing antifungal armamentariums has become a severe threat to global health. There is also concern regarding increased rates of azole resistance in Aspergillus fumigatus and Terbinafine resistance in Trichophyton species. To overcome this concern of resistance to regular therapies, new antifungal drugs with novel and effective mechanisms are crucially needed. Herbal remedies may be promising strategies for the treatment of resistant infections. We aimed to investigate the in vitro and silico activity of piperlongumine against clinical azole susceptible/resistant A. fumigatus and terbinafine-susceptible/resistant Trichophyton species. In the current study, piperlongumine demonstrated potent antifungal activity, with minimum inhibitory concentrations (MICs) ranging from 0.016-4 μg/mL against Trichophyton isolates and 0.25-2 μg/mL for A. fumigatus isolates. Additionally, molecular docking studies indicated that piperlongumine has a strong binding affinity to the active sites of squalene epoxidase and sterol 14-alpha demethylase. However, further studies are warranted to correlate these findings with clinical outcomes and provide the basis for further investigations to pave the way for developing novel antifungal agents.

Synergy of Histone Acetyltransferase Inhibitor (HATi) with Quercetin Inhibits Biofilm Formation in Candida tropicalis.

Histone acetyltransferase inhibitors (HATi) are mechanism-based inhibitors that show promise in the treatment of several illnesses, including diabetes, hyperlipidemia, cancer, and Alzheimer's disease. The work emphasizes the significance of HATi as a possible treatment strategy against Candida species biofilms. Here, in this study, we found that combining a HATi, anacardic acid, and quercetin, a known flavonoid, significantly prevented biofilm formation by C. tropicalis. We further show that C. tropicalis exhibited a considerable downregulation of drug-resistance gene expression (CDR1 and MDR1) when co-administrated. Additionally, in silico studies revealed that the anacardic acid (AA) interacts strongly with a histone acetyltransferase, Rtt109, which may account for the observed biofilm inhibitory effect. In conclusion, the study illustrates how HATi may be used to potentiate the inhibitory action of phytoactives or antifungals against drug-resistant yeast infections.

Models for Inducing Experimental Cryptococcosis in Mice.

Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.

Investigation of the first cluster of Candida auris cases among pediatric patients in the United States―Nevada, May 2022

Background:Candida auris is a frequently drug-resistant yeast that can cause invasive disease and is easily transmitted in healthcare settings. Pediatric cases are rare in the United States, with <10 reported before 2022. In August 2021, the first C. auris case in Las Vegas was identified in an adult. By May 2022, 117 cases were identified across 16 healthcare facilities, including 3 pediatric cases at an acute-care hospital (ACH) with adult cases, representing the first pediatric cluster in the United States. The CDC and Nevada Division of Public and Behavioral Health (NVDPBH) sought to describe these cases and risk factors for C. auris acquisition. Methods: We defined a case as a patient’s first positive C. auris specimen. We reviewed medical records and infection prevention and control (IPC) practices. Environmental sampling was conducted on high-touch surfaces throughout affected adult and pediatric units. Isolate relatedness was assessed using whole-genome sequencing (WGS). Results: All 3 pediatric patients were born at the facility and had congenital heart defects. All were aged <6 months when they developed C. auris bloodstream infections; 2 developed C. auris endocarditis. One patient died. Patients overlapped in the pediatric cardiac intensive care unit; 2 did not leave between birth and C. auris infection. Mobile medical equipment was shared between adult and pediatric patients; lapses in cleaning and disinfection of shared mobile medical equipment and environmental surfaces were observed, presenting opportunities for transmission. Overall, 32 environmental samples were collected, and C. auris was isolated from 2 specimens from an adult unit without current cases. One was a composite sample from an adult patient’s bed handles, railings, tray table and call buttons, and the second was from an adult lift-assistance device. WGS of specimens from adult and pediatric cases and environmental isolates were in the same genetic cluster, with 2–10 single-nucleotide polymorphisms (SNPs) different, supporting within-hospital transmission. The pediatric cases varied by 0–3 SNPs; at least 2 were highly related. Conclusions:C. auris was likely introduced to the pediatric population from adults via inadequately cleaned and disinfected mobile medical equipment. We made recommendations to ensure adequate cleaning and disinfection and implement monitoring and audits. No pediatric cases have been identified since. This investigation demonstrates transmission can occur between unrelated units and populations and that robust infection prevention and control practices throughout the facility are critical for reducing C. auris environmental burden and limiting transmission, including to previously unaffected vulnerable populations, like children.Disclosures: None

Combined antibiofilm activity of synthetic peptides and antifungal drugs against Candida spp.

Introduction: Candida krusei and Candida albicans are biofilm-forming drug-resistant yeasts that cause bloodstream infections that can lead to death. Materials & methods: nystatin and itraconazole were combined with two synthetic peptides, PepGAT and PepKAA, to evaluate the synergistic effect against Candida biofilms. Additionally, scanning electron and fluorescence microscopies were employed to understand the mechanism behind the synergistic activity. Results: Peptides enhanced the action of drugs to inhibit the biofilm formation of C. krusei and C. albicans and the degradation of mature biofilms of C. krusei. In combination with antifungal drugs, peptides'mechanism of action involved cell wall and membrane damageand overproduction of reactive oxygen species. Additionally, in combination, the peptides reduced the toxicity of drugs to red blood cells. Conclusion: These results reveal that the synthetic peptides enhanced the antibiofilm activity of drugs, in addition to reducing their toxicity. Thus, these peptides have strong potential as adjuvants and to decrease the toxicity of drugs.

935. A Hospital Cluster of the Emerging Drug-resistant Yeast Saprochaete clavata

Background Saprochaete clavata, an ascomycetous yeast intrinsically resistant to echinocandins, is a rare yet emerging pathogen associated with invasive infections in immunosuppressed patients, particularly those with haematological malignancies. It is commonly misidentified as the closely-related S. capitata. Outbreaks have been associated with a high mortality rate of >50%, due in part to delayed diagnosis and resistance to commonly-used antifungals. Environmental source identification is challenging, although dishwashers and milk flasks have been implicated in previous hospital clusters.MethodsWe describe a cluster of five haematology-oncology patients with disseminated S. clavata infections between April 2020 and April 2021 following current or recent admissions to the same ward. ResultsAll had prolonged (median=24 days, range=9-210) and profound immunosuppression from chemotherapy and/or stem cell transplantation for acute myeloid leukaemia (n=3) or lymphoma (n=2) at the time of culture positivity. Four were severely neutropaenic (median=0.08/mm3, range=0.01-0.26). Median patient age was 62 years (range=58-73). S. clavata was isolated from blood (n=3), urine (n=2), and liver tissue (n=1) samples. Whole genome sequencing of these isolates was performed to confirm the presence of an outbreak. All patients received empirical treatment with intravenous caspofungin before culture-guided therapy with intravenous liposomal amphotericin B +/- oral flucytosine. Two of the five patients died although both had advanced refractory malignancy. Detailed environmental sampling of fridges/freezers, drains, and vents in patient rooms and clean areas for handling or storage of food and medication failed to identify a clear point source despite isolation of multiple environmental organisms. No further cases have emerged after intensification of the cleaning regimen in these areas.ConclusionOur experience highlights the emerging threat of drug-resistant yeasts particularly in the immunocompromised. Management of such outbreaks requires a multidisciplinary approach incorporating antifungal stewardship, infection control, and environmental microbiology, alongside close clinical liaison between haemato-oncologists and infection specialists.Disclosures All Authors: No reported disclosures

E-test Determination of Antifungal Susceptibility of Candida Species Isolated from Turkeys.

IntroductionCandida species are a natural component of the intestinal tract microflora, but in favourable conditions they can cause superficial, mucosal, or even systemic candidiasis. Poultry production might be a source of human drug-resistant yeast infections, including Candida spp. The limited data concerning the antifungal susceptibility of poultry Candida isolates prompted us to carry out research to determine the susceptibility of isolates from turkey intestinal tracts.Method and MaterialsThe beak cavity, crop and cloaca were swabbed of 580 turkeys from 58 flocks in western Poland. The susceptibility tests were conducted using the E-test method with amphotericin B, fluconazole, itraconazole, and voriconazole on 52 isolates of C. albicans, C. catenulata, C. glabrata, C. palmioleophila, C. rugosa, C. krusei and C. lusitaniae.ResultsAll isolates were susceptible to voriconazole. According to the MIC values obtained for amphotericin B and fluconazole, all Candida spp. isolates were classified as susceptible according to the described breakpoints except for C. krusei, which was the only isolate that was amphotericin B-, fluconazole- and itraconazole-resistant. The susceptibility to itraconazole varied: 11 of the Candida isolates were susceptible (21.1%), 29 were dose-dependently susceptible (55.8%), and 12 isolates were resistant (23.1%).ConclusionThere are few resistant strains of Candida in turkeys, and the drug resistance varies. When Candida passes from turkeys to humans, there is a wide range of antifungal treatment options.

Virulence and in vitro antifungal susceptibility of Candida albicans and Candida catenulata from laying hens

In spite of evidence that domestic and wild birds may act as carriers of human pathogenic fungi, data on the role of laying hens as reservoirs of drug resistant and virulent yeasts is lacking. Here, we assess several virulence factors (phospholipase and haemolysin activity) and the antifungal susceptibility profiles of 84 Candida albicans and 17 Candida catenulata strains isolated from cloacae (group A), faeces (group B) and eggs (group C) of laying hens. Of these strains, 95% C. albicans and 23% C. catenulata strains displayed phospholipase and haemolytic activities. For C. albicans, the highest values of phospholipase (Pz = 0.62) and haemolytic activities (Hz = 0.49) were recorded among the strains from group C whilst for C. catenulata (Pz = 0.54; Hz = 0.49) among those from group A. High minimum inhibitory concentration (MIC) values for azoles and amphotericin B (AmB) were recorded irrespective of their sources in all C. albicans strains. A total of 22 C. albicans strains were multidrug resistant, displaying resistance to fluconazole, itraconazole (ITZ), voriconazole (VOR) and posaconazole (POS). All C. catenulata strains from group C were resistant to ITZ, POS, micafungin and anidulafungin and susceptible to AmB. In this study, C. albicans and C. catenulata isolated from the cloacae, faeces and eggs of laying hens produced phospholipase and haemolysin and might be multidrug resistant. In the environment (faeces) or in eggs, C. albicans and C. catenulata strains might acquire pathogenic virulence traits and/or show multidrug resistance profiles. Based on these results, breeding and handling of laying hens and/or eggs may have implications for human and animal health.

The Effect of Ten Essential Oils on Several Cutaneous Drug-Resistant Microorganisms and Their Cyto/Genotoxic and Antioxidant Properties.

In this study, we determined the antimicrobial activity of ten essential oils (EOs)—oregano, thyme, clove, arborvitae, cassia, lemongrass, melaleuca, eucalyptus, lavender, and clary sage—against drug-resistant microorganisms previously isolated from patients with skin infections. The essential oil compositions were determined using gas chromatography coupled to mass spectrometry (GC/MS). The assayed bacteria included Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter koseri, and Klebsiella pneumoniae. Two drug-resistant yeasts (Candida albicans and Candida parapsilosis) were also involved in our survey. Oregano, thyme, cassia, lemongrass and arborvitae showed very strong antibacterial and antifungal activity against all tested strains. These results show that these essential oils may be effective in preventing the growth of the drug-resistant microorganisms responsible for wound infections. In this study, the genotoxic effects of tested essential oils on healthy human keratinocytes HaCaT were evaluated using the comet assay for the first time. These results revealed that none of the essential oils induced significant DNA damage in vitro after 24 h. Moreover, the treatment of HaCaT cells with essential oils increased the total antioxidant status (TAS) level. The obtained results indicate that EOs could be used as a potential source of safe and potent natural antimicrobial and antioxidant agents in the pharmaceutical and food industries.

The antiproliferative peptide Ctn[15-34] is active against multidrug-resistant yeasts Candida albicans and Cryptococcus neoformans.

Crotalicidin (Ctn), a cathelicidin-related antimicrobial peptide from the South American rattlesnake venom gland, and its C-terminal Ctn[15-34] fragment, have exhibited important activities against micro-organisms, trypanosomatid protozoa and certain lines of tumour cells. Herein, the activity against clinical strains of fluconazole-resistant Candida albicans and of amphotericin B and fluconazole-resistant Cryptococcus neoformans was investigated. Microdilution and luminescent cell viability tests were used to evaluate and compare the susceptibility of pathogenic yeasts to these peptides. The time-kill curves of the most active Ctn[15-34] alone or in combination with fluconazole against drug-resistant yeasts were determined. Concomitantly, the fungicidal and/or fungistatic effects of Ctn[15-34] were visualized by the spotting test. The peptides were active against all strains, including those resistant to antifungal agents. The association of fluconazole with both Ctn and Ctn[15-34], although not synergic, was additive. In contrast, such pattern was not observed for C. neoformans. Overall, Ctn and Ctn[15-34] are potential antifungal leads displaying anti-yeast activities against clinical isolates endowed with drug resistance mechanisms. The effective peptide activity against resistant strains of pathogenic yeasts demonstrates that crotalicidin-derived peptides are promising templates to develop new antifungal pharmaceuticals.

Diagnosis, management and prevention of Candida auris in hospitals: position statement of the Australasian Society for Infectious Diseases.

Candida auris is an emerging drug-resistant yeast responsible for hospital outbreaks. This statement reviews the evidence regarding diagnosis, treatment and prevention of this organism and provides consensus recommendations for clinicians and microbiologists in Australia and New Zealand. C. auris has been isolated in over 30 countries (including Australia). Bloodstream infections are the most frequently reported infections. Infections have crude mortality of 30-60%. Acquisition is generally healthcare-associated and risks include underlying chronic disease, immunocompromise and presence of indwelling medical devices. C. auris may be misidentified by conventional phenotypic methods. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry or sequencing of the internal transcribed spacer regions and/or the D1/D2 regions of the 28S ribosomal DNA are therefore required for definitive laboratory identification. Antifungal drug resistance, particularly to fluconazole, is common, with variable resistance to amphotericin B and echinocandins. Echinocandins are currently recommended as first-line therapy for infection in adults and children ≥2 months of age. For neonates and infants <2 months of age, amphotericin B deoxycholate is recommended. Healthcare facilities with C. auris should implement a multimodal control response. Colonised or infected patients should be isolated in single rooms with Standard and Contact Precautions. Close contacts, patients transferred from facilities with endemic C. auris or admitted following stay in overseas healthcare institutions should be pre-emptively isolated and screened for colonisation. Composite swabs of the axilla and groin should be collected. Routine screening of healthcare workers and the environment is not recommended. Detergents and sporicidal disinfectants should be used for environmental decontamination.

Type 2 diabetes mellitus and oral Candida colonization: Analysis of risk factors in a Sri Lankan cohort

Aims: Oral candidiasis is a major oral manifestation of uncontrolled diabetes mellitus, and a number of cofactors are associated with the pathogenesis of this infection. Here, we describe the prevalence of oral Candida in a Sri Lankan cohort of type 2 diabetes mellitus and risk factors that predispose them to this common fungal infection.Methods: A case–control study was conducted in 250 diabetics with type 2 diabetes and 81 nondiabetic controls. Clinical and demographic data were collected using an interviewer administered questionnaire, and patient records. Oral rinse samples were collected to determine the candidal carriage, and the resultant yeast growth was quantified and speciated using multiplex-PCR and phenotypic analyses. Chi-square test (χ2 test) and Fisher exact test were used for the determination of the significant relationships between risk factors and oral candidiasis.Results: The oral prevalence of Candida species among both groups was similar (81%) although a significantly higher proportion of diabetics (32.8%) yielded >2000 CFU/mL of yeasts compared with only 12.3% of the healthy controls (p < .05). Significant associations were noted between oral candidal carriage amongst diabetics, and (i) denture wearing, (ii) female gender and (iii) cigarette smoking (all, p < .05). Amongst both groups, C.albicans was the most common Candida species isolated followed by C. parapsilosis, C. tropicalis and C. glabrata.Conclusions: The oral infestation of Candida in our Sri Lankan cohort of diabetics is significantly higher than their healthy counterparts, and co-carriage of multiple yeast species is a common finding in the study population. As there are no previous such reports of the latter phenomenon particularly from the Asian region it is noteworthy, mainly in view of the recent data on the emergence of drug-resistant yeast species the world over.

Phospholipid biosynthesis disruption renders the yeast cells sensitive to antifungals.

To understand the role of phospholipids on Cdr1p (drug exporter)-mediated drug resistance in yeast, the phospholipids biosynthesis genes PSD1, PSD2, CHO2, and OPI3 were deleted in a strain of Saccharomyces cerevisiae already overexpressing Cdr1-GFP of Candida albicans as a heterologous system. The effect of phospholipids biosynthesis gene deletion was analyzed on Cdr1p-GFP-mediated drug resistance as well as its localization. The results indicate that phospholipids biosynthesis disruption makes the cell sensitive to several drugs including fluconazole (FLC), with Δpsd1/Cdr1-GFP being worst affected. Interestingly, unlike sterols and sphingolipids, the localization of Cdr1p was unaffected by phospholipid biosynthesis gene disruption. Concomitantly, phospholipids mutants also showed an increase in reactive oxygen species (ROS) generation, as verified by fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method. In addition, the sensitivity of phospholipid mutants with FLC was found to be synergistic to ROS generation, resulting in further reduction of growth. Thus, this study proposes phospholipid biosynthesis as a novel target for antifungal therapy.

Rapid and extensive karyotype diversification in haploid clinical Candida auris isolates

Candida auris is a newly emerged pathogenic microbe, having been identified as a medically relevant fungus as recently as 2009. It is one of the most drug-resistant yeast species known to date and its emergence and population structure are unusual. Because of its recent emergence, we are largely ignorant about fundamental aspects of its general biology, life cycle, and population dynamics. Here, we report the karyotype variability of 26 C. auris strains representing the four main clades. We demonstrate that all strains are haploid and have a highly plastic karyotype containing five to seven chromosomes, which can undergo marked alterations within a short time frame when the fungus is put under genotoxic, heat, or osmotic stress. No simple correlation was found between karyotype pattern, drug resistance, and clade affiliation indicating that karyotype heterogeneity is rapidly evolving. As with other Candida species, these marked karyotype differences between isolates are likely to have an important impact on pathogenic traits of C. auris.

LB13. Candida auris in NYC: A Health System’s Experience Treating the Emerging Drug-Resistant Yeast

Background Candida auris is emerging multidrug-resistant yeast that can cause serious infections with published mortality rates as high as 60%. It was first recognized in 2009 and has been reported in over a dozen countries. The current United States outbreak was identified in 2016 with New York City (NYC) as the epicenter. The aim of this evaluation was to describe the clinical infections and outcomes with C. auris in a large health system in NYC.MethodsCases were identified from clinical specimens collected December 2015–June 2018 from the Mount Sinai Hospital Clinical Microbiology Laboratory, the central laboratory for the Mount Sinai Health System, which encompasses seven hospitals across NYC. All C. auris isolates were confirmed by the New York State Department of Health Wadsworth Center. Medical charts were reviewed. A case was included if C. auris grew from a sterile body site, an antifungal treatment was initiated or the patient expired before the yeast was identified on Gram stain.ResultsTwenty-nine possible cases were identified with 23 meeting the case definition. These cases included 19 bloodstream infections (BSI), two intra-abdominal abscesses, one skin soft tissue infection, and one otitis externa. Using the MIC breakpoints recommended by the Centers for Disease Control and Prevention, 100% of isolates tested were susceptible to caspofungin, 29% were susceptible to amphotericin B, and 17% were susceptible to fluconazole. Nineteen patients received antifungal treatment, 13 with caspofungin monotherapy and four with sequential therapy of caspofungin followed by an azole (three with fluconazole, one with posaconazole). Fifteen (65%) patients expired within 90 days of the positive culture. Fourteen of the deaths were in candidemic patients, despite that eight (57%) of these patients had documented microbiologic clearance after appropriate therapy. The 90-day mortality rate was 74% for BSI.ConclusionsThis case series is the largest reported in the United States. Candidemia was the most common site of infection and had a very high 90-day mortality rate, despite sterilization of the blood. These findings highlight the significant morbidity and mortality associated with C. auris and the need to focus efforts on rapid diagnostics and infection prevention.Disclosures All authors: No reported disclosures.

Penetrating cations induce pleiotropic drug resistance in yeast

Substrates of pleiotropic drug resistance (PDR) transporters can induce the expression of corresponding transporter genes by binding to their transcription factors. Penetrating cations are substrates of PDR transporters and theoretically may also activate the expression of transporter genes. However, the accumulation of penetrating cations inside mitochondria may prevent the sensing of these molecules. Thus, whether penetrating cations induce PDR is unclear. Using Saccharomyces cerevisiae as a model, we studied the effects of penetrating cations on the activation of PDR. We found that the lipophilic cation dodecyltriphenylphosphonium (C12TPP) induced the expression of the plasma membrane PDR transporter genes PDR5, SNQ2 and YOR1. Moreover, a 1-hour incubation with C12TPP increased the concentration of Pdr5p and Snq2p and prevented the accumulation of the PDR transporter substrate Nile red. The transcription factor PDR1 was required to mediate these effects, while PDR3 was dispensable. The deletion of the YAP1 or RTG2 genes encoding components of the mitochondria-to-nucleus signalling pathway did not prevent the C12TPP-induced increase in Pdr5-GFP. Taken together, our data suggest (i) that the sequestration of lipophilic cations inside mitochondria does not significantly inhibit sensing by PDR activators and (ii) that the activation mechanisms do not require mitochondria as a signalling module.

Genome Sequence of the Amphotericin B-Resistant Candida duobushaemulonii Strain B09383.

ABSTRACTCandida duobushaemulonii is a drug-resistant yeast that can cause invasive candidiasis. Here, we report the first genome sequence of C. duobushaemulonii, isolate B09383, generated using PacBio sequencing technology. The estimated genome size was 12.5 Mb with a GC content of 46.84%.

Molecular basis of antifungal drug resistance in yeasts

Besides inherent differences in in vitro susceptibilities, clinically-relevant yeast species may acquire resistance upon exposure to most antifungal drugs used in the clinic. In recent years, major fundamental research studies have been conducted to improve our understanding of the molecular basis of antifungal resistance. This topic is of major interest as antifungal resistance in yeast is clearly evolving and is correlated with clinical failure. This minireview is an overview of the most recent findings about key molecular mechanisms evolving in human pathogenic yeasts, particularly Candida spp., in the context of antifungal drug resistance. Also included are the methods currently available for in vitro antifungal susceptibility testing and for molecular detection of mutations associated with resistance. Finally, the genetic drivers of antifungal resistance are discussed in light of the spectra of multidrug resistance as observed in Candida glabrata.

Fruits as the vehicle of drug resistant pathogenic yeasts

We investigated the diversity and drug susceptibility of pathogenic yeasts on fruit surfaces. Fruits were purchased from supermarkets and washed with buffer. The pellets were re-suspended in medium after centrifugation. The cell suspensions were plated onto CHROMagar Candida medium. Yeasts were identified by ribosomal DNA sequencing and their drug susceptibilities were determined by broth microdilution assay. Of 184 isolates, comprised of 55 species, from 22 different types of fruits, 29 species, including Candida famata, Candida fermentati, Candida guilliermondii, Candida intermedia, Candida krusei, Candida orthopsilosis, Candida parapsilosis, Candida pelliculosa, Candida tropicalis, and others have been reported to cause diseases in humans. In addition to C. krusei, intrinsically resistant to fluconazole, all Rhodotorula and Rhodosporidium species were resistant to fluconazole. One each of C. tropicalis isolate was belonged to diploid sequence type (DST)149 and DST225, genotypes also detected in isolates from humans. Furthermore, the DST225 isolate was less susceptible to azole drugs. The susceptibilities to azole drugs for clinical and agricultural usage were associated to each other. It is important to be aware of the existence of pathogenic yeasts, especially drug-resistant ones, on the fruit surfaces, a potential route for pathogenic yeasts to be transmitted to humans.

The Small RNA GcvB Promotes Mutagenic Break Repair by Opposing the Membrane Stress Response.

ABSTRACTMicrobes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli, repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σS) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σS response, achieved via opposing the membrane stress (σE) response. Cells that lack gcvB were MBR deficient and displayed reduced σS-dependent transcription but not reduced σS protein levels. The defects in MBR and σS-dependent transcription in ΔgcvB cells were alleviated by artificially increasing σS levels, implying that GcvB promotes mutagenesis by allowing a normal σS response. ΔgcvB cells were highly induced for the σE response, and blocking σE response induction restored both mutagenesis and σS-promoted transcription. We suggest that GcvB may promote the σS response and mutagenesis indirectly, by promoting membrane integrity, which keeps σE levels lower. At high levels, σE might outcompete σS for binding RNA polymerase and so reduce the σS response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis.IMPORTANCE Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli. The roles of many sRNAs in E. coli remain unknown. This study shows that ΔgcvB cells, which lack the GcvB sRNA, display a hyperactivated membrane stress response and reduced general stress response, possibly because of sigma factor competition for RNA polymerase. This results in a mutagenic break repair defect. The data illuminate a function of GcvB sRNA in opposing the membrane stress response, and thus indirectly upregulating mutagenesis.