Whole-genome sequencing confirms a persistent candidaemia clonal outbreak due to multidrug-resistant Candida parapsilosis.

OBJECTIVES: Although perceived as a rare clinical entity, recent studies have noted the emergence of MDR C. parapsilosis (MDR-Cp) isolates from single patients (resistant to both azole and echinocandins). We previously reported a case series of MDR-Cp isolates carrying a novel FKS1R658G mutation. Herein, we identified an echinocandin-naive patient infected with MDR-Cp a few months after the previously described isolates. WGS and CRISPR-Cas9 editing were used to explore the origin of the new MDR-Cp isolates, and to determine if the novel mutation confers echinocandin resistance. METHODS: WGS was applied to assess the clonality of these isolates and CRISPR-Cas9 editing and a Galleria mellonella model were used to examine whether FKS1R658G confers echinocandin resistance. RESULTS: Fluconazole treatment failed, and the patient was successfully treated with liposomal amphotericin B (LAMB). WGS proved that all historical and novel MDR-Cp strains were clonal and distant from the fluconazole-resistant outbreak cluster in the same hospital. CRISPR-Cas9 editing and G. mellonella virulence assays confirmed that FKS1R658G confers echinocandin resistance in vitro and in vivo. Interestingly, the FKS1R658G mutant showed a very modest fitness cost compared with the parental WT strain, consistent with the persistence of the MDR-Cp cluster in our hospital. CONCLUSIONS: Our study showcases the emergence of MDR-Cp isolates as a novel threat in clinical settings, which undermines the efficacy of the two most widely used antifungal drugs against candidiasis, leaving only LAMB as a last resort. Additionally, surveillance studies and WGS are warranted to effectively establish infection control and antifungal stewardship strategies.

SeqEditor: an application for primer design and sequence analysis with or without GTF/GFF files.

MOTIVATION: Sequence analyses oriented to investigate specific features, patterns and functions of protein and DNA/RNA sequences usually require tools based on graphic interfaces whose main characteristic is their intuitiveness and interactivity with the user's expertise, especially when curation or primer design tasks are required. However, interface-based tools usually pose certain computational limitations when managing large sequences or complex datasets, such as genome and transcriptome assemblies. Having these requirments in mind we have developed SeqEditor an interactive software tool for nucleotide and protein sequences' analysis. RESULT: SeqEditor is a cross-platform desktop application for the analysis of nucleotide and protein sequences. It is managed through a Graphical User Interface and can work either as a graphical sequence browser or as a fasta task manager for multi-fasta files. SeqEditor has been optimized for the management of large sequences, such as contigs, scaffolds or even chromosomes, and includes a GTF/GFF viewer to visualize and manage annotation files. In turn, this allows for content mining from reference genomes and transcriptomes with similar efficiency to that of command line tools. SeqEditor also incorporates a set of tools for singleplex and multiplex PCR primer design and pooling that uses a newly optimized and validated search strategy for target and species-specific primers. All these features make SeqEditor a flexible application that can be used to analyses complex sequences, design primers in PCR assays oriented for diagnosis, and/or manage, edit and personalize reference sequence datasets. AVAILABILITYAND IMPLEMENTATION: SeqEditor was developed in Java using Eclipse Rich Client Platform and is publicly available at https://gpro.biotechvana.com/download/SeqEditor as binaries for Windows, Linux and Mac OS. The user manual and tutorials are available online at https://gpro.biotechvana.com/tool/seqeditor/manual. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetically related micafungin-resistant Candida parapsilosis blood isolates harbouring novel mutation R658G in hotspot 1 of Fks1p: a new challenge?

BACKGROUND: Echinocandin resistance rarely occurs in clinical Candida parapsilosis isolates and the underlying mechanism is unknown. OBJECTIVES: To determine the prevalence of echinocandin resistance and the underlying mechanism for a large collection of C. parapsilosis blood isolates and to determine whether the echinocandin-resistant isolates were clonally related. METHODS: C. parapsilosis blood isolates (n = 213) were subjected to antifungal susceptibility testing (CLSI M27), for micafungin, anidulafungin, amphotericin B and, if appropriate, caspofungin. Hotspot (HS) 1 and HS2 of FKS1 were sequenced for all isolates (n = 213) and microsatellite typing was performed for echinocandin-resistant isolates. RESULTS: All isolates were susceptible to amphotericin B and two isolates were intermediate to anidulafungin (MIC = 4 mg/L), while micafungin resistance was noted in four isolates (MIC >8 mg/L); three of which were also fluconazole resistant and therefore were MDR. Interestingly, micafungin-resistant isolates, but not those intermediate to anidulafungin, carried novel mutation R658G in HS1 of Fks1p; three of which also harboured Y132F+K143R in Erg11. The first isolate (MICR1) was recovered in November 2017 from a patient admitted to paediatric gastroenterology who showed therapeutic failure under caspofungin treatment. MICR2-MICR4 were collected during 2018-19 and were recovered from three echinocandin-naive paediatric-surgery patients; the isolates shared the same genotype. CONCLUSIONS: Herein, for the first time (to the best of our knowledge), we identified micafungin-resistant C. parapsilosis blood isolates harbouring a novel mutation in HS1 of FKS1, which was likely attributable to in vitro micafungin resistance and in vivo caspofungin therapeutic failure. The acquisition of micafungin-resistant C. parapsilosis isolates in echinocandin-naive patients likely implicates clonal expansion, as supported by the close genetic relatedness of MICR2-MICR4.

Epidemiology of candidemia in Shiraz, southern Iran: A prospective multicenter study (2016-2018).

Systematic candidemia studies, especially in southern Iran, are scarce. In the current prospective study, we investigated candidemia in three major healthcare centers of Shiraz, the largest city in southern Iran. Yeast isolates from blood and other sterile body fluids were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method. Clinical data were retrieved from patients' medical records. In total, 113 yeast isolates were recovered from 109 patients, over 60% of whom received fluconazole. Antifungal drugs were prescribed without considering species identification or AFST. The all-cause mortality rate was 28%. Almost 30% of the patients were from intensive care units (ICUs). Candida albicans (56/113; 49.5%) was the most prevalent species followed by C. glabrata (26/113; 23%), C. parapsilosis (13/113; 11.5%), C. tropicalis (7/113; 6.2%), and C. dubliniensis (5/113; 4.4%). Only five isolates showed antifungal resistance or decreased susceptibility to fluconazole: one C. orthopsilosis isolate from an azole-naïve patient and two C. glabrata, one C. albicans, and one C. dubliniensis isolates from patients treated with azoles, who developed therapeutic failure against azoles later. Our results revealed a low level of antifungal resistance but a notable rate of azole therapeutic failure among patients with candidemia due to non-albicans Candida species, which threaten the efficacy of fluconazole, the most widely used antifungal in southern regions of Iran. Candidemia studies should not be confined to ICUs and treatment should be administered based on species identification and AFST results.

Landscape of candidemia is blurred in Iran, and only two studies from Tehran have extensively explored the epidemiology of candidemia. However, candidemia data from the other regions are notoriously scarce, which precludes from reaching a consensus regarding species distribution, the burden of antifungal resistance, and the clinical features of infected patients. Therefore, we conducted the current prospective candidemia study in Shiraz, one of the largest cities located in the south of Iran, from April 2016 to April 2018. More than 63% of the candidemia infections were treated by fluconazole and species identification and antifungal susceptibility testing were not used for decision making regarding the choice of antifungal treatment. Approximately 70% of the candidemia cases occurred in the wards outside of the ICUs. Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis were the five leading causative agents of candidemia. Antifungal resistance was rare and fluconazole resistance and/or non-wild type phenotypes were noticed in five isolates, only one was C. albicans and the rest were non-albicans Candida (NAC) species, including C. glabrata, C. dubliniensis, and C. orthopsilosis. Except for C. orthopsilosis, which was isolated from an azole-naïve patient, the rest of isolates were recovered from patients treated with azoles and all showed therapeutic failure to azoles. Collectively, our data will complete the candidemia picture in Iran and show that, although the level of resistance was rare, the therapeutic failure was notable among NAC species, which threatens the efficacy of fluconazole, the most widely used antifungal in Southern regions of Iran. Moreover, we showed that candidemia is poorly managed in Iran since species identification tools along with antifungal susceptibility testing were not used to select appropriate antifungal treatment.

First Report of Candidemia Clonal Outbreak Caused by Emerging Fluconazole-Resistant Candida parapsilosis Isolates Harboring Y132F and/or Y132F+K143R in Turkey.

Clonal outbreaks of fluconazole-resistant (FLZR) Candida parapsilosis isolates have been reported in several countries. Despite its being the second leading cause of candidemia, the azole resistance mechanisms and the clonal expansion of FLZR C. parapsilosis blood isolates have not been reported in Turkey. In this study, we consecutively collected C. parapsilosis blood isolates (n = 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced ERG11 for all and MRR1, TAC1, and UPC2 for a selected number of C. parapsilosis isolates. The typing resolution of two widely used techniques, amplified fragment length polymorphism typing (AFLP) and microsatellite typing (MST), and the biofilm production of FLZR isolates with and without Y132F were compared. Approximately 27% of isolates were FLZR (60/225), among which 90% (54/60) harbored known mutations in Erg11, including Y132F (24/60) and Y132F+K143R (19/60). Several mutations specific to FLZR isolates were found in MRR1, TAC1, and UPC2 AFLP grouped isolates into two clusters, while MST revealed several clusters. The majority of Y132F/Y132F+K143R isolates grouped in clonal clusters, which significantly expanded throughout 2007 to 2019 in neonatal wards. Candida parapsilosis isolates carrying Y132F were associated with significantly higher mortality and less biofilm production than other FLZR isolates. Collectively, we documented the first outbreak of FLZR C. parapsilosis blood isolates in Turkey. The MRR1, TAC1, and UPC2 mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in C. parapsilosis MST should be a preferred method for clonal analysis of C. parapsilosis isolates in outbreak scenarios.

Clinical and microbiological features of candiduria in critically ill adult patients in Shiraz, Iran (2016-2018): deviations from international guidelines and fluconazole therapeutic failure.

Candiduria is common among patients admitted to intensive care units (ICUs); however, clinical and microbiological data are limited, which accounts for non-compliance with international guidelines, including over treatment of asymptomatic candiduria that promotes antifungal resistance. This prospective study included adult patients admitted to ICUs of five referral hospitals in Shiraz, Iran, during 2016-2018. Species were identified by MALDI-TOF MS, and antifungal susceptibility was assessed according to CLSI M27-A3/S4. Among 2086 patients, 162 and 293 developed candiduria and bacteriuria, respectively. In total, 174 yeast isolates were collected; 88.5% were Candida albicans (91/174; 52.2%), C. glabrata (38/174; 21.8%), and C. tropicalis (25/174; 14.3%). Antifungal resistance was rare; only two isolates (one C. tropicalis and one C. krusei) were fluconazole resistant. Symptomatic candiduria was noted in 31.4% of patients (51/162); only 37% (19/51) of them were treated and 36.82% (7/19) showed fluconazole therapeutic failure. Two symptomatic patients developed candidemia shortly after candiduria. Among asymptomatic patients, 31.5% (35/111) were overtreated with fluconazole. The mortality rate was 25.3% (41/162); it did not differ between symptomatic and asymptomatic patients. Our results indicate that deviation from standard-of-care treatment for candiduria is a matter of concern given the high rate of fluconazole therapeutic failure among patients with symptomatic candiduria. LAY SUMMARY: Candiduria is an underestimated clinical presentation among critically ill patients and detailed data are scarce in this regard. Given the high rate of fluconazole therapeutic failure and development of candidemia in some cases, the mistreatment of candiduria should not be overlooked by clinicians.

Antifungal susceptibility, genotyping, resistance mechanism, and clinical profile of Candida tropicalis blood isolates.

Candida tropicalis is one of the major candidaemia agents, associated with the highest mortality rates among Candida species, and developing resistance to azoles. Little is known about the molecular mechanisms of azole resistance, genotypic diversity, and the clinical background of C. tropicalis infections. Consequently, this study was designed to address those questions. Sixty-four C. tropicalis bloodstream isolates from 62 patients from three cities in Iran (2014-2019) were analyzed. Strain identification, antifungal susceptibility testing, and genotypic diversity analysis were performed by MALDI-TOF MS, CLSI-M27 A3/S4 protocol, and amplified fragment length polymorphism (AFLP) fingerprinting, respectively. Genes related to drug resistance (ERG11, MRR1, TAC1, UPC2, and FKS1 hotspot9s) were sequenced. The overall mortality rate was 59.6% (37/62). Strains were resistant to micafungin [minimum inhibitory concentration (MIC) ≥1 µg/ml, 2/64], itraconazole (MIC > 0.5 µg/ml, 2/64), fluconazole (FLZ; MIC ≥ 8 µg/ml, 4/64), and voriconazole (MIC ≥ 1 µg/ml, 7/64). Pan-azole and FLZ + VRZ resistance were observed in one and two isolates, respectively, while none of the patients were exposed to azoles. MRR1 (T255P, 647S), TAC1 (N164I, R47Q), and UPC2 (T241A, Q340H, T381S) mutations were exclusively identified in FLZ-resistant isolates. AFLP fingerprinting revealed five major and seven minor genotypes; genotype G4 was predominant in all centers. The increasing number of FLZ-R C. tropicalis blood isolates and acquiring FLZ-R in FLZ-naive patients limit the efficiency of FLZ, especially in developing countries. The high mortality rate warrants reaching a consensus regarding the nosocomial mode of C. tropicalis transmission.

Low level of antifungal resistance of Candida glabrata blood isolates in Turkey: Fluconazole minimum inhibitory concentration and FKS mutations can predict therapeutic failure.

BACKGROUND: Candida glabrata is the third leading cause of candidaemia in Turkey; however, the data regarding antifungal resistance mechanisms and genotypic diversity in association with their clinical implication are limited. OBJECTIVES: To assess genotypic diversity, antifungal susceptibility and mechanisms of drug resistance of C glabrata blood isolates and their association with patients' outcome in a retrospective multicentre study. PATIENTS/METHODS: Isolates from 107 patients were identified by ITS sequencing and analysed by multilocus microsatellite typing, antifungal susceptibility testing, and sequencing of PDR1 and FKS1/2 hotspots (HSs). RESULTS: Candida glabrata prevalence in Ege University Hospital was twofold higher in 2014-2019 than in 2005-2014. Six of the analysed isolates had fluconazole MICs ≥ 32 µg/mL; of them, five harboured unique PDR1 mutations. Although echinocandin resistance was not detected, three isolates had mutations in HS1-Fks1 (S629T, n = 1) and HS1-Fks2 (S663P, n = 2); one of the latter was also fluconazole-resistant. All patients infected with isolates carrying HS-FKS mutations and/or demonstrating fluconazole MIC ≥ 32 µg/mL (except one without clinical data) showed therapeutic failure (TF) with echinocandin and fluconazole; seven such isolates were collected in Ege (n = 4) and Gulhane (n = 3) hospitals and six detected recently. Among 34 identified genotypes, none were associated with mortality or enriched for fluconazole-resistant isolates. CONCLUSION: Antifungal susceptibility testing should be supplemented with HS-FKS sequencing to predict TF for echinocandins, whereas fluconazole MIC ≥ 32 µg/mL may predict TF. Recent emergence of C glabrata isolates associated with antifungal TF warrants future comprehensive prospective studies in Turkey.

Low Level of Antifungal Resistance in Iranian Isolates of Candida glabrata Recovered from Blood Samples in a Multicenter Study from 2015 to 2018 and Potential Prognostic Values of Genotyping and Sequencing of PDR1.

Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candidaglabrata derived from patients suffering from candidemia. C. glabrata isolates were retrospectively collected from four major cities in Iran; identified by a 21-plex PCR, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and large subunit of ribosomal DNA sequencing; and genotyped by amplified fragment length polymorphism (AFLP). Mutations in PDR1, ERG11, and hot spot 1 (HS1) of FKS1 and FKS2 were investigated, and antifungal susceptibility testing (AFST) was performed (by the CLSI M27-A3 and M27-S4 methods). Seventy isolates of C. glabrata were collected from 65 patients with a median age of 58 years. Fluconazole was the most widely used (29.23%) and least effective antifungal agent. The overall crude mortality rate was 35.4%. Only one strain was resistant to fluconazole, and 57.7% and 37.5% of the isolates were non-wild type (non-WT) for susceptibility to caspofungin and voriconazole, respectively. All isolates showed the WT phenotype for amphotericin B, posaconazole, and itraconazole. HS1 of FKS1 and FKS2 did not harbor any mutations, while numerous missense mutations were observed in PDR1 and ERG11 AFLP clustered our isolates into nine genotypes; among them, genotypes 1 and 2 were significantly associated with a higher mortality rate (P = 0.034 and P = 0.022, α < 0.05). Moreover, 83.3% of patients infected with strains harboring a single new mutation in PDR1, T745A, died despite treatment with fluconazole or caspofungin. Overall, Iranian isolates of C. glabrata were susceptible to the major antifungal drugs. Application of genotyping techniques and sequencing of a specific gene (PDR1) might have prognostic implications.

Unequivocal identification of an underestimated opportunistic yeast species, Cyberlindnera fabianii, and its close relatives using a dual-function PCR and literature review of published cases.

Although Cyberlindnera fabinaii is a rare opportunist yeast species, its ability to cause septicemia, produce biofilm, and rapid acquisition of resistance to fluconazole and voriconazole, reinforced the urge for its identification from its closely related species. Widely used biochemical assays mainly identify Cyberlindnera fabinaii as Cyberlindnera jadinii and Wickerhamomyces anomalus, resulting in underestimation of this yeast in clinical settings. Moreover, the urge for a reliable molecular means of identification remains unsolved for 28 years. In order to unequivocally differentiate Cy. fabianii, Cy. mississipiensis, Cy. jadinii, and W. anomalus, we designed a dual-function multiplex polymerase chain reaction (PCR) assay. Challenging our dual-function multiplex PCR assay with 30 most clinically important yeast species, proved its specificity. Although conventional PCR could differentiate four target species, the real-time PCR counterpart due to Tm overlap misidentified Cy. mississipiensis as Cy. jadinii. Alongside of presenting a comprehensive literature review of published cases of Cy. fabianii from 1990 to 2018, we collected various clinical isolates from Tehran, Shiraz, and Fasa (July 1, 2017, to December 31, 2017) to find a passive relative distribution of these closely-related species in Iran. Subjecting our Iranian collection of yeast isolates to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and LSU and ITS rDNA sequencing revealed six isolates of Cy. fabianii (central venous catheter n = 2 and vaginal swabs n = 4) and one isolate of Cy. jadinii (vaginal swabs). Due to the use of biochemical assays in global ARTEMIS study, we encourage reidentification of clinical isolates of Cy. jadinii and Cy. jadinii using MALDI-TOF or Sanger sequencing that might lead to correcting the distribution of this fungus.

Echinocandin persistence directly impacts the evolution of resistance and survival of the pathogenic fungus Candida glabrata.

Recent epidemiological studies documented an alarming increase in the prevalence of echinocandin-resistant (ECR) Candida glabrata blood isolates. ECR isolates are known to arise from a minor subpopulation of a clonal population, termed echinocandin persisters. Although it is believed that isolates with a higher echinocandin persistence (ECP) are more likely to develop ECR, the implication of ECP needs to be better understood. Moreover, replacing laborious and time-consuming traditional approaches to determine ECP levels with rapid, convenient, and reliable tools is imperative to advance our understanding of this emerging concept in clinical practice. Herein, using extensive ex vivo and in vivo systemic infection models, we showed that high ECP isolates are less effectively cleared by micafungin treatment and exclusively give rise to ECR colonies. Additionally, we developed a flow cytometry-based tool that takes advantage of a SYTOX-based assay for the stratification of ECP levels. Once challenged with various collections of echinocandin-susceptible blood isolates, our assay reliably differentiated ECP levels in vitro and predicted ECP levels in real time under ex vivo and in vivo conditions when compared to traditional methods relying on colony-forming unit counting. Given the high and low ECP predictive values of 92.3% and 82.3%, respectively, our assay showed a high agreement with traditional approach. Collectively, our study supports the concept of ECP level determination in clinical settings and provides a robust tool scalable for high-throughput settings. Application of this tool facilitates the interrogation of mutant and drug libraries to further our understanding of persister biology and designing anti-persister therapeutics. IMPORTANCE: Candida glabrata is a prevalent fungal pathogen able to replicate inside macrophages and rapidly develop resistance against frontline antifungal echinocandins. Multiple studies have shown that echinocandin resistance is fueled by the survival of a small subpopulation of susceptible cells surviving lethal concentrations of echinocandins. Importantly, bacterial pathogens that exhibit high antibiotic persistence also impose a high burden and generate more antibiotic-resistant colonies. Nonetheless, the implications of echinocandin persistence (ECP) among the clinical isolates of C. glabrata have not been defined. Additionally, ECP level determination relies on a laborious and time-consuming method, which is prone to high variation. By exploiting in vivo systemic infection and ex vivo models, we showed that C. glabrata isolates with a higher ECP are associated with a higher burden and more likely develop echinocandin resistance upon micafungin treatment. Additionally, we developed an assay that reliably determines ECP levels in real time. Therefore, our study identified C. glabrata isolates displaying high ECP levels as important entities and provided a reliable and convenient tool for measuring echinocandin persistence, which is extendable to other fungal and bacterial pathogens.

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches.

Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance.

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Candida parapsilosis isolates carrying mutations outside FKS1 hotspot regions confer high echinocandin tolerance and facilitate the development of echinocandin resistance.

Candida parapsilosis is a significant cause of candidemia worldwide. Echinocandin-resistant (ECR) and echinocandin-tolerant (ECT) C. parapsilosis isolates have been reported in various countries but are rare. Resistance and tolerance are predominantly caused by mutations related to the hotspot (HS) regions of the FKS1 gene. A relatively high proportion of clinical C. parapsilosis isolates carrying mutations outside the HS regions has been noted in some studies, but an association with echinocandin (EC) resistance or tolerance was not explored. Herein, CRISPR-Cas9 was used and the association between amino acid substitution in FKS1 outside HS 1/2 (V595I, S745L, M1328I, F1386S, and A1422G) with EC susceptibility profile was delineated. None of the mutations conferred EC resistance, but they resulted in a significantly higher level of EC tolerance than the parental isolate, ATCC 22019. When incubated on agar plates containing ECs, specifically caspofungin and micafungin, ECR colonies were exclusively observed among ECT isolates, particularly mutants carrying V595I, S745L, and F1386S. Additionally, mutants had significantly better growth rates in yeast extract peptone dextrose (YPD) and YPD containing agents inducing membrane and oxidative stresses. The mutants had a trivial fitness cost in the Galleria mellonella model relative to ATCC 22019. Collectively, this study supports epidemiological studies to catalog mutations occurring outside the HS regions of FKS1, even if they do not confer EC resistance. These mutations are important as they potentially confer a higher level of EC tolerance and a higher propensity to develop EC resistance, therefore unveiling a novel mechanism of EC tolerance in C. parapsilosis. The identification of EC tolerance in C. parapsilosis may have direct clinical benefit in patient management.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

IMPORTANCE: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Worldwide emergence of fluconazole-resistant Candida parapsilosis: current framework and future research roadmap.

Candida parapsilosis is one of the most commen causes of life-threatening candidaemia, particularly in premature neonates, individuals with cancer of the haematopoietic system, and recipients of organ transplants. Historically, drug-susceptible strains have been linked to clonal outbreaks. However, worldwide studies started since 2018 have reported severe outbreaks among adults caused by fluconazole-resistant strains. Outbreaks caused by fluconazole-resistant strains are associated with high mortality rates and can persist despite strict infection control strategies. The emergence of resistance threatens the efficacy of azoles, which is the most widely used class of antifungals and the only available oral treatment option for candidaemia. The fact that most patients infected with fluconazole-resistant strains are azole-naive underscores the high potential adaptability of fluconazole-resistant strains to diverse hosts, environmental niches, and reservoirs. Another concern is the multidrug-resistant and echinocandin-tolerant C parapsilosis isolates, which emerged in 2020. Raising awareness, establishing effective clinical interventions, and understanding the biology and pathogenesis of fluconazole-resistant C parapsilosis are urgently needed to improve treatment strategies and outcomes.

Candidemia among Hospitalized Pediatric Patients Caused by Several Clonal Lineages of Candida parapsilosis.

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

Candidemia Among Coronavirus Disease 2019 Patients in Turkey Admitted to Intensive Care Units: A Retrospective Multicenter Study.

Background: We evaluated the epidemiology of candidemia among coronavirus disease 2019 (COVID-19) patients admitted to intensive care units (ICUs). Methods: We conducted a retrospective multicenter study in Turkey between April and December 2020. Results: Twenty-eight of 148 enrolled patients developed candidemia, yielding an incidence of 19% and incidence rate of 14/1000 patient-days. The probability of acquiring candidemia at 10, 20, and 30 days of ICU admission was 6%, 26%, and 50%, respectively. More than 80% of patients received antibiotics, corticosteroid, and mechanical ventilation. Receipt of a carbapenem (odds ratio [OR] = 6.0, 95% confidence interval [CI] = 1.6-22.3, P = .008), central venous catheter (OR = 4.3, 95% CI = 1.3-14.2, P = .02), and bacteremia preceding candidemia (OR = 6.6, 95% CI = 2.1-20.1, P = .001) were independent risk factors for candidemia. The mortality rate did not differ between patients with and without candidemia. Age (OR = 1.05, 95% CI = 1.01-1.09, P = .02) and mechanical ventilation (OR = 61, 95% CI = 15.8-234.9, P < .0001) were independent risk factors for death. Candida albicans was the most prevalent species overall. In Izmir, Candida parapsilosis accounted for 50% (2 of 4) of candidemia. Both C parapsilosis isolates were fluconazole nonsusceptible, harbored Erg11-Y132F mutation, and were clonal based on whole-genome sequencing. The 2 infected patients resided in ICUs with ongoing outbreaks due to fluconazole-resistant C parapsilosis. Conclusions: Physicians should be aware of the elevated risk for candidemia among patients with COVID-19 who require ICU care. Prolonged ICU exposure and ICU practices rendered to COVID-19 patients are important contributing factors to candidemia. Emphasis should be placed on (1) heightened infection control in the ICU and (2) developing antibiotic stewardship strategies to reduce irrational antimicrobial therapy.