Fatal co-infection by multiple pathogens in an indigenous woman with autoimmune hemolytic anemia and tuberculosis: a case report.

BACKGROUND: Tuberculosis (TB), one of the leading causes of death worldwide, has a higher incidence among indigenous people. Albeit uncommon, autoimmune hemolytic anemia (AIHA) has been deemed a risk condition to develop mycobacterial infection, as a result of the immunosuppressive treatments. TB, in turn, can be a predisposing factor for secondary infections. CASE PRESENTATION: Here we present a case of a 28-year-old indigenous woman from Colombia, previously diagnosed with AIHA and pulmonary TB. Despite various treatments, therapies and medical interventions, the patient died after severe medullary aplasia of multiple causes, including secondary myelotoxicity by immunosuppressive therapy and secondary disseminated infections, underlining infection by Staphylococcus aureus, Klebsiella pneumoniae and Candida glabrata, which were identified as drug-resistant microorganisms. Together, this led to significant clinical complications. Invasive aspergillosis was diagnosed at autopsy. CONCLUSIONS: This report presents a rarely finding of AIHA followed by TB, and highlights the great challenges of dealing with co-infections, particularly by drug resistant pathogens. It also aims to spur governments and public health authorities to focus attention in the prevention, screening and management of TB, especially among vulnerable communities, such as indigenous people.

Investigation of adhesion status of Candida species to the surface of resin materials produced at different angles with additive manufacturing.

BACKGROUND: The aim of this study was to evaluate the adhesion of Candida glabrata, Candida albicans, Candida krusei, Candida parapsilosis and Candida tropicalis yeasts to disk-shaped resin materials produced from resin which used in the production of surgical guide with 0, 45 and 90-degrees printing orientations by Liquid Crystal Display additive manufacturing technology. METHODS: Disk-shaped specimens were printed with surgical guide resin using the Liquid Crystal Display production technique in 3 printing orientations (0, 45 and 90-degrees). Surface roughness and contact angle values were evaluated. Real-Time PCR analysis was performed to evaluate Candida adhesion (C. glabrata, C. albicans, C. krusei, C. parapsilosis and C. tropicalis) Field emission scanning electron microscope (FESEM) images of the materials were obtained. RESULTS: Specimens oriented at 45-degrees demonstrated higher surface roughness (P < .05) and lower contact angle values than other groups. No significant difference was found in the adhesion of C. glabrata, C. albicans, and C. parapsilosis among specimens printed at 0, 45, and 90-degrees orientations (P > .05). A higher proportion of C. krusei and C. tropicalis was found in the specimens printed at orientation degrees of 45 = 90 < 0 with statistical significance. Analyzing the adhesion of all Candida species reveals no statistical disparity among the printing orientations. CONCLUSIONS: The surface roughness, contact angle, and adhesion of certain Candida species are affected by printing orientations. Hence, careful consideration of the printing orientation is crucial for fabricating products with desirable properties. In 45-degree production, roughness increases due to the layered production forming steps, whereas in 0-degree production, certain Candida species exhibit high adhesion due to the formation of porous structures. Consequently, considering these factors, it is advisable to opt for production at 90-degrees, while also considering other anticipated characteristics.

Candida albicans and Candida glabrata: global priority pathogens.

SUMMARYA significant increase in the incidence of Candida-mediated infections has been observed in the last decade, mainly due to rising numbers of susceptible individuals. Recently, the World Health Organization published its first fungal pathogen priority list, with Candida species listed in medium, high, and critical priority categories. This review is a synthesis of information and recent advances in our understanding of two of these species-Candida albicans and Candida glabrata. Of these, C. albicans is the most common cause of candidemia around the world and is categorized as a critical priority pathogen. C. glabrata is considered a high-priority pathogen and has become an increasingly important cause of candidemia in recent years. It is now the second most common causative agent of candidemia in many geographical regions. Despite their differences and phylogenetic divergence, they are successful as pathogens and commensals of humans. Both species can cause a broad variety of infections, ranging from superficial to potentially lethal systemic infections. While they share similarities in certain infection strategies, including tissue adhesion and invasion, they differ significantly in key aspects of their biology, interaction with immune cells, host damage strategies, and metabolic adaptations. Here we provide insights on key aspects of their biology, epidemiology, commensal and pathogenic lifestyles, interactions with the immune system, and antifungal resistance.

Candida glabrata (Nakaseomyces glabrata): A systematic review of clinical and microbiological data from 2011 to 2021 to inform the World Health Organization Fungal Priority Pathogens List.

Recognising the growing global burden of fungal infections, the World Health Organization (WHO) established an advisory group consisting of experts in fungal diseases to develop a Fungal Priority Pathogen List. Pathogens were ranked based on their research and development needs and perceived public health importance using a series of global surveys and pathogen characteristics derived from systematic reviews. This systematic review evaluates the features and global impact of invasive disease caused by Candida glabrata (Nakaseomyces glabrata). PubMed and Web of Science were searched for studies reporting on mortality, morbidity (hospitalization and disability), drug resistance (including isolates from sterile and non-sterile sites, since these reflect the same organisms causing invasive infections), preventability, yearly incidence, diagnostics, treatability, and distribution/emergence in the last 10 years. Candida glabrata (N. glabrata) causes difficult-to-treat invasive infections, particularly in patients with underlying conditions such as immunodeficiency, diabetes, or those who have received broad-spectrum antibiotics or chemotherapy. Beyond standard infection prevention and control measures, no specific preventative measures have been described. We found that infection is associated with high mortality rates and that there is a lack of data on complications and sequelae. Resistance to azoles is common and well described in echinocandins-in both cases, the resistance rates are increasing. Candida glabrata remains mostly susceptible to amphotericin and flucytosine. However, the incidence of the disease is increasing, both at the population level and as a proportion of all invasive yeast infections, and the increases appear related to the use of antifungal agents.

Complement receptor 3-dependent engagement by Candida glabrata ß-glucan modulates dendritic cells to induce regulatory T-cell expansion.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

Phytolacca tetramera berries extracts and its main constituents as potentiators of antifungal drugs against Candida spp.

BACKGROUND: Extensive antifungal drug use has enhanced fungal resistance, resulting in persistent mycoses. Combining antifungal plant extracts/compounds with these drugs offers good alternatives to increase the activity of both partners, minimize side effects, and overcome drug resistance. In our previous study, Phytolacca tetramera berries extracts demonstrated activity against Candida spp., correlating with the amount of the main constituent phytolaccoside B and its genin, phytolaccagenin. The extracts and phytolaccagenin altered the fungal plasma membrane by binding to ergosterol, whereas phytolaccoside B increased chitin synthase activity. However, the presence of triterpenoid saponins in Phytolacca spp. has been linked to acute toxicity in humans. PURPOSE: This study aimed to evaluate combinations of P. tetramera berries extracts, phytolaccoside B and phytolaccagenin, together with commercial antifungals [amphotericin B, fluconazole, itraconazole, posaconazole, and caspofungin] against Candida albicans and Candida glabrata, to find synergistic effects with multi-target actions, in which the doses of both partners are reduced, and therefore their toxicity. Additionally, we intended to explore their anti-virulence capacity, thereby hindering the development of drug-resistant strains. METHODS: The effects of these combinations were evaluated using both the checkerboard and isobologram methods. Fractional Inhibitory Concentration Index and Dose Reduction Index were calculated to interpret the combination results. To confirm the multi-target effect, studies on mechanisms of action of synergistic mixtures were performed using ergosterol-binding and quantification assays. The ability to inhibit Candida virulence factors, including biofilm formation and eradication from inert surfaces, was also evaluated. Quantification of active markers was performed using a validated UHPLC-ESI-MS method. RESULTS: Eight synergistic combinations of P. tetramera extracts or phytolaccagenin (but not phytolaccoside B) with itraconazole or posaconazole were obtained against C. albicans, including a resistant strain. These mixtures acted by binding to ergosterol, decreasing its whole content, and inhibiting Candida biofilm formation in 96-well microplates and feeding tubes in vitro, but were unable to eradicate preformed biofilms. CONCLUSIONS: This study demonstrated the synergistic and anti-virulence effects of P. tetramera berries extracts and phytolaccagenin with antifungal drugs against Candida spp., providing novel treatment avenues for fungal infections with reduced doses of both natural products and commercial antifungals, thereby mitigating potential human toxicity concerns.

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis. IMPORTANCE: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata.

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.

Antimicrobial and anti-biofilm properties of oleuropein against Escherichia coli and fluconazole-resistant isolates of Candida albicans and Candida glabrata.

BACKGROUND: Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level. MATERIALS AND METHODS: In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method. RESULTS: The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p < 0.0001). The anti-biofilm effect of oleuropein was confirmed by SEM. RT-qPCR indicated significant down regulation of expression genes involved in biofilm formation in Candida albicans (Hwp1, Als3) and Candida glabrata (Epa1, Epa6) as well as Escherichia coli (LuxS, Pfs) genes after culture with a MIC/2 of oleuropein (p < 0.0001). CONCLUSIONS: The results indicate that oleuropein has antifungal and antibacterial properties that enable it to inhibit or destroy the formation of fungal and bacterial biofilm.

Adhesive and biofilm-forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins.

BACKGROUND: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. OBJECTIVES: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. METHODS: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. RESULTS: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. CONCLUSIONS: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

The Hog1 MAPK substrate governs Candida glabrata-epithelial cell adhesion via the histone H2A variant.

CgHog1, terminal kinase of the high-osmolarity glycerol signalling pathway, orchestrates cellular response to multiple external stimuli including surplus-environmental iron in the human fungal pathogen Candida glabrata (Cg). However, CgHog1 substrates remain unidentified. Here, we show that CgHog1 adversely affects Cg adherence to host stomach and kidney epithelial cells in vitro, but promotes Cg survival in the iron-rich gastrointestinal tract niche. Further, CgHog1 interactome and in vitro phosphorylation analysis revealed CgSub2 (putative RNA helicase) to be a CgHog1 substrate, with CgSub2 also governing iron homeostasis and host adhesion. CgSub2 positively regulated EPA1 (encodes a major adhesin) expression and host adherence via its interactor CgHtz1 (histone H2A variant). Notably, both CgHog1 and surplus environmental iron had a negative impact on CgSub2-CgHtz1 interaction, with CgHTZ1 or CgSUB2 deletion reversing the elevated adherence of Cghog1Δ to epithelial cells. Finally, the surplus-extracellular iron led to CgHog1 activation, increased CgSub2 phosphorylation, elevated CgSub2-CgHta (canonical histone H2A) interaction, and EPA1 transcriptional activation, thereby underscoring the iron-responsive, CgHog1-induced exchange of histone partners of CgSub2. Altogether, our work mechanistically defines how CgHog1 couples Epa1 adhesin expression with iron abundance, and point towards specific chromatin composition modification programs that probably aid fungal pathogens align their adherence to iron-rich (gut) and iron-poor (blood) host niches.

Molecular Epidemiology and Antifungal Susceptibility Profile in Nakaseomyces glabrata Species Complex: A 5-Year Countrywide Study.

BACKGROUND: The current study aimed to identify Iranian Nakaseomyces (Candida) glabrata complex species in the clinical isolates and determine their antifungal susceptibility profile. METHODS: In total, 320 N. glabrata clinical isolates were collected from patients hospitalized in different geographical regions of Iran. The initial screening was performed by morphological characteristics on CHROMagar Candida. Each isolate was identified by targeting the D1/D2 rDNA using a multiplex-PCR method. To validate the mPCR method and determine genetic diversity, the ITS-rDNA region was randomly sequenced in 40 isolates. Additionally, antifungal susceptibility was evaluated against nine antifungal agents following the CLSI M27-A4 guidelines. RESULTS: All clinical isolates from Iran were identified as N. glabrata. The analysis of ITS-rDNA sequence data revealed the presence of eight distinct ITS clades and 10 haplotypes among the 40 isolates of N. glabrata. The predominant clades identified were Clades VII, V, and IV, which respectively accounted for 22.5%, 17.5%, and 17.5% isolates. The widest MIC ranges were observed for voriconazole (0.016-8 µg/mL) and isavuconazole (0.016-2 µg/mL), whereas the narrowest ranges were seen with itraconazole and amphotericin B (0.25-2 µg/mL). CONCLUSION: Haplotype diversity can be a valuable approach for studying the genetic diversity, transmission patterns, and epidemiology of the N. glabrata complex.

Impact of glucocorticoids and rapamycin on autophagy in Candida glabrata-infected macrophages from BALB/c mice.

Objective: In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in the elimination process through the formation of a single-membrane phagosome known as LC3-associated phagocytosis (LAP). Building on this, we propose the hypothesis that glucocorticoids may hinder macrophage phagocytosis of Candida glabrata by suppressing LAP, and rapamycin could potentially reverse this inhibitory effect. Methods: RAW264.7 cells were employed for investigating the immune response to Candida glabrata infection. Various reagents, including dexamethasone, rapamycin, and specific antibodies, were utilized in experimental setups. Assays, such as fluorescence microscopy, flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay), Western blot, and confocal microscopy, were conducted to assess phagocytosis, cytokine levels, protein expression, viability, and autophagy dynamics. Results: Glucocorticoids significantly inhibited macrophage autophagy, impairing the cells' ability to combat Candida glabrata. Conversely, rapamycin exhibited a dual role, initially inhibiting and subsequently promoting phagocytosis of Candida glabrata by macrophages. Glucocorticoids hinder macrophage autophagy in Candida glabrata infection by suppressing the MTOR pathway(mammalian target of rapamycin pathway), while the activation of MTOR pathway by Candida glabrata diminishes over time. Conclusion: Our study elucidates the intricate interplay between glucocorticoids, rapamycin, and macrophage autophagy during Candida glabrata infection. Understanding the implications of these interactions not only sheds light on the host immune response dynamics but also unveils potential therapeutic avenues for managing fungal infections.

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy.

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (≥99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Analysis of candidemia cases in a city hospital during the COVID-19 pandemic.

OBJECTIVE: The frequency and mortality of candidemia remain important. Non-albicans Candida species such as C. auris are increasing. PATIENTS AND METHODS: A retrospective review of adult patients diagnosed with bloodstream infection due to Candida species in the 17 months between July 1, 2020, and December 1, 2021, was performed. Yeast colonies grown in culture were identified by matrix-assisted laser desorption/ionization time-of-flight. Antifungal susceptibility tests of Candida strains were performed with Sensititre YeastOne (TREK Diagnostic Systems Inc., Westlake, Ohio) kits, and minimum inhibitory concentration values were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints. RESULTS: In total, 217 patients (mean age 64.9±15.7 years) were included. C. albicans was the most common fungus (detected in 82 patients; 37.8%), followed by C. parapsilosis (17.1%), C. glabrata (15.2%), C. tropicalis (15.2%), and C. auris (9%). Candidemia developed in 175 (81.4%) of the cases during their intensive care unit stay. Fluconazole (41.0%) and caspofungin (36.4%) were the two most frequently used antifungal agents in antifungal therapy. There were 114 (52.3%) deaths in the study group. Mortality rates were found to be lower in patients infected with C. parapsilosis or C. auris. Age and previous COVID-19 infection were other important risk factors. When the 217 Candida spp. were examined, resistance and intermediate susceptibility results were higher when EUCAST criteria were used. While the two methods were found to be fully compatible only for fluconazole, a partial agreement was also observed for voriconazole. CONCLUSIONS: As our study observed, the COVID-19 pandemic brought increasing numbers of immunosuppressed patients, widespread use of antibacterials, and central venous catheters, increasing the frequency and mortality of candidemia cases. All health institutions should be prepared for the diagnosis and treatment of candidemia. In addition, C. auris, the frequency of which has increased in recent years, is a new factor that should be considered in candidemia cases.

Silver vanadate nanomaterial incorporated into heat-cured resin and coating in printed resin - Antimicrobial activity in two multi-species biofilms and wettability.

OBJECTIVES: To incorporate the nanostructured silver vanadate decorated with silver nanoparticles (AgVO3) into denture base materials: heat-cured (HC) and 3D printed (3DP) resins, at concentrations of 2.5 %, 5 %, and 10 %; and to evaluate the antimicrobial activity in two multi-species biofilm: (1) Candida albicans, Candida glabrata, and Streptococcus mutans, (2) Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus, and the wettability. METHODS: The AgVO3 was added to the HC powder, and printed samples were coated with 3DP with AgVO3 incorporated. After biofilm formation, the antimicrobial activity was evaluated by colony forming units per milliliter (CFU/mL), metabolic activity, and epifluorescence microscopy. Wettability was assessed by the contact angles with water and artificial saliva. RESULTS: In biofilm (1), HC-5 % and HC-10 % showed activity against S. mutans, HC-10 % against C. glabrata, and HC-10 % and 3DP-10 % had higher CFU/mL of C. albicans. 3DP-5 % had lower metabolic activity than the 3DP control. In biofilm (2), HC-10 % reduced S. aureus and P. aeruginosa, and HC-5 %, 3DP-2.5 %, and 3DP-5 % reduced S. aureus. 3DP incorporated with AgVO3, HC-5 %, and HC-10 % reduced biofilm (2) metabolic activity. 3DP-5 % and 3DP-10 % increased wettability with water and saliva. CONCLUSION: HC-10 % was effective against C. glabrata, S. mutans, P. aeruginosa, and S. aureus, and HC-5 % reduced S. mutans and S. aureus. For 3DP, 2.5 % and 5 % reduced S. aureus. The incorporation of AgVO3 into both resins reduced the metabolic activity of biofilms but had no effect on C. albicans. The wettability of the 3DP with water and saliva increased with the addition of AgVO3. CLINICAL SIGNIFICANCE: The incorporation of silver vanadate into the denture base materials provides antimicrobial efficacy and can prevent the aggravation of oral and systemic diseases. The incorporation of nanomaterials into printed resins is challenging and the coating is an alternative to obtain the inner denture base with antimicrobial effect.

Microevolution of Candida glabrata (Nakaseomyces glabrata) during an infection.

Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas ß-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains. These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata.

BACKGROUND: Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. RESULTS: Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. CONCLUSIONS: The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

The Zanthoxylum armatum fruit’s oil exterminates Candida cells by inhibiting ergosterol biosynthesis without generating reactive oxygen species

Candida spp. is a significant cause of topical and fungal infections in humans. In addition to Candida albicans, many non-albicans species such as C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii cause severe infections. The main antifungal agents belong to three different classes, including azoles, polyenes, and echinocandins. However, resistance to all three categories of drugs has been reported. Therefore, there is an urgent need to search for other alternatives with antifungal activity. Many herbal extracts and compounds from natural sources show excellent antifungal activity. In this study, we used an oil extract from the fruits of Zanthoxylum armatum, which showed significant antifungal activity against various Candida spp. by two different methods—minimum inhibitory concentration (MIC) and agar diffusion. In addition, we attempted to explore the possible mechanism of action in C. albicans. It was found that the antifungal activity of Z. armatum oil is fungicidal and involves a decrease in the level of ergosterol in the cell membrane. The decrease in ergosterol level resulted in increased passive diffusion of a fluorescent molecule, rhodamine6G, across the plasma membrane, indicating increased membrane fluidity. The oil-treated cells showed decreased germ tube formation, an important indicator of C. albicans’ virulence. The fungal cells also exhibited decreased attachment to the buccal epithelium, the first step toward invasion, biofilm formation, and damage to oral epithelial cells. Interestingly, unlike most antifungal agents, in which the generation of reactive oxygen species is responsible for killing, no significant effect was observed in the present study. (AU)

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

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