The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p < 0.0001), while no significant differences were observed in the expression levels of the MDR1 and CDR1 genes. The minimum inhibitory concentration of nisin against C. tropicalis ranged from 2 to 8 μg/mL. Nisin treatment inhibited the growth of azole-resistant C. tropicalis, with over a four-fold reduction in OD600 nm values observed at the 8-h time point, while it promoted the transition of C. tropicalis from the spore phase to the hyphal phase, as observed on cryo-scanning electron microscopy. The results of biofilm quantification using crystal violet staining indicated a significant decrease in OD570 nm values in the nisin-treated group compared to the controls (p < 0.0001). Among the 21 azole-resistant C. tropicalis strains, the biofilm formation was inhibited in 17 strains (17/21, 81%), and more than 85% inhibition of biofilm formation was observed in the representative strains. With regard to the molecular mechanisms, the expression of the BCR1 and UPC2 genes in the azole-resistant strains was down-regulated on nisin treatment (p < 0.05). In conclusion, we demonstrated, for the first time, that nisin has antifungal activity and significant anti-biofilm activity against clinical isolates of azole-resistant C. tropicalis strains. Based on the findings, nisin could be a promising alternative antifungal agent for combating azole-resistant C. tropicalis infections.

Azoles are the primary antifungal drugs used to treat infections caused by Aspergillus fumigatus. However, the emergence of azole resistance in A. fumigatus has become a global health concern despite the low proportion of resistant isolates in natural populations. In bacteria, antibiotic resistance incurs a fitness cost that renders strains less competitive in the absence of antibiotics. Consequently, fitness cost is a key determinant of the spread of resistant mutations. However, the cost of azole resistance and its underlying causes in A. fumigatus remain poorly understood. In this observation, we revealed that the 10 out of 15 screened azole-resistant isolates, which possessed the most common azole-targeted cyp51A mutations, particularly the presence of tandem repeats in the promoter region, exhibit fitness cost when competing with the susceptible isolates in azole-free environments. These results suggest that fitness cost may significantly influence the dynamics of azole resistance, which ultimately contributes to the low prevalence of azole-resistant A. fumigatus isolates in the environment and clinic. By constructing in situ cyp51A mutations in a parental azole-susceptible strain and reintroducing the wild-type cyp51A gene into the azole-resistant strains, we demonstrated that fitness cost is not directly dependent on cyp51A mutations but is instead associated with the evolution of variable mutations related to conidial germination or other unknown development-related processes. Importantly, our observations unexpectedly revealed that some azole-resistant isolates showed no detectable fitness cost, and some even exhibited significantly increased competitive fitness in azole-free environments, highlighting the potential risk associated with the prevalence of these isolates.
OBJECTIVE: Azole resistance in the human fungal pathogen Aspergillus fumigatus presents a global public health challenge. Understanding the epidemic trends and evolutionary patterns of azole resistance is critical to prevent and control the spread of azole-resistant isolates. The primary cause is the mutation of the drug target 14α-sterol-demethylase Cyp51A, yet its impact on competitive ability remains uncertain. Our competition assays revealed a diverse range of fitness outcomes for environmental and clinical cyp51A-mutated isolates. We have shown that this fitness cost is not reliant on cyp51A mutations but might be linked to unknown mutations induced by stress conditions. Among these isolates, the majority displayed fitness costs, while a few displayed enhanced competitive ability, which may have a potential risk of spread and the need to closely monitor these isolates. Our observation reveals the variation in fitness costs among azole-resistant isolates of A. fumigatus, highlighting the significant role of fitness cost in the spread of resistant strains.

UNASSIGNED: The objective of this study was to determine the clinical distribution, in vitro antifungal susceptibility and underlying resistance mechanisms of Aspergillus fumigatus (A. fumigatus) isolates from the General Hospital of Ningxia Medical University between November 2021 and May 2023.
UNASSIGNED: Antifungal susceptibility testing was performed using the Sensititre YeastOne YO10, and isolates with high minimal inhibitory concentrations (MICs) were further confirmed using the standard broth microdilution assays established by the Clinical and Laboratory Standards Institute (CLSI) M38-third edition. Whole-Genome Resequencing and RT-qPCR in azole-resistant A. fumigatus strains were performed to investigate the underlying resistance mechanisms.
UNASSIGNED: Overall, a total of 276 A. fumigatus isolates were identified from various clinical departments, showing an increasing trend in the number of isolates over the past 3 years. Two azole-resistant A. fumigatus strains (0.72%) were observed, one of which showed overexpression of cyp51A, cyp51B, cdr1B, MDR1/2, artR, srbA, erg24A, and erg4B, but no cyp51A mutation. However, the other strain harbored two alterations in the cyp51A sequences (L98H/S297T). Therefore, we first described two azole-resistant clinical A. fumigatus strains in Ningxia, China, and reported one azole-resistant strain that has the L98H/S297T mutations in the cyp51A gene without any tandem repeat (TR) sequences in the promoter region.
UNASSIGNED: This study emphasizes the importance of enhancing attention and surveillance of azole-resistant A. fumigatus, particularly those with non-TR point mutations of cyp51A or non-cyp51A mutations, in order to gain a better understanding of their prevalence and spread in the region.

Aspergillus fumigatus, a prevalent saprophytic fungus in the atmosphere, is known to rapidly induce severe invasive aspergillosis (IA) upon inhalation of its conidia by humans or animals. The mortality rate associated with IA exceeds 50%. The misuse of antifungal agents has contributed to the emergence of numerous highly pathogenic drug-resistant strains of A. fumigatus. Our study found that the combination of domiphen and itraconazole had sound synergistic antimicrobial effects against wild-type and itraconazole-resistant A. fumigatus in vivo and in vitro through MIC, FIC, plate inoculation, growth curve experiments, and Galleria mellonella infection model. Drug cytotoxicity and pharmacological tests for acute toxicity assays demonstrated that both itraconazole and domiphen showed minimal cytotoxicity and good biocompatibility. The transcriptome sequencing experiment demonstrated that domiphen exerted a suppressive effect on the expression of various genes, including those involved in drug efflux, redox regulation, and cellular membrane and cell wall remodeling. The present investigation explores the synergistic antimicrobial mechanisms of domiphen and itraconazole, encompassing three key aspects: (i) domiphen inhibited the efflux of itraconazole by reducing the expression of drug efflux-related genes, (ii) the combination has good ability to disrupt the cell membrane and cell wall, (iii) the combination also can remove biofilm more effectively. In summary, the utilization of domiphen as a synergist of itraconazole exhibited disruptive effects on the biofilm, cell wall, and cell membrane of A. fumigatus. This subsequently led to a modified distribution of itraconazole within the fungal organism, ultimately resulting in enhanced antifungal efficacy. The results of this study may provide a new therapeutic strategy for the treatment of IA caused by drug-resistant A. fumigatus.

Aspergillus fumigatus is a common causative pathogen of aspergillosis. At present, triazole resistance of A. fumigatus poses an important challenge to human health globally. In this study, the biological characteristics and mechanisms of azole resistance of five A. fumigatus strains (AF1, AF2, AF4, AF5, and AF8) were explored. There were notable differences in the sporulation and biofilm formation abilities of the five test strains as compared to the standard strain AF293. The ability of strain AF1 to avoid phagocytosis by MH-S cells was significantly decreased as compared to strain AF293, while that of strains AF2, AF4, and AF5 were significantly increased. Fungal burden analysis with Galleria mellonella larvae revealed differences in pathogenicity among the five strains. Moreover, the broth microdilution and E-test assays confirmed that strains AF1 and AF2 were resistant to itraconazole and isaconazole, while strains AF4, AF5, and AF8 were resistant to voriconazole and isaconazole. Strains AF1 and AF2 carried the cyp51A mutations TR34/L98H/V242I/S297T/F495I combined with the hmg1 mutation S541G, whereas strains AF4 and AF8 carried the cyp51A mutation TR46/Y121F/V242I/T289A, while strain AF5 had no cyp51A mutation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed differences in the expression levels of genes associated with ergosterol synthesis and efflux pumps among the five strains. In addition, transcriptomics, RT-qPCR, and the NAD+/NADH ratio demonstrated that the mechanism of voriconazole resistance of strain AF5 was related to overexpression of genes associated with energy production and efflux pumps. These findings will help to further elucidate the triazole resistance mechanism in A. fumigatus.

Azole resistance in the human fungal pathogen Aspergillus fumigatus is becoming a major threat to global health. To date, mutations in the azole target-encoding cyp51A gene have been implicated in conferring azole resistance, but a steady increase in the number of A. fumigatus isolates with azole resistance resulting from non-cyp51A mutations has been recognized. Previous studies have revealed that some isolates with non-cyp51A mutation-induced azole resistance are related to mitochondrial dysfunction. However, knowledge of the molecular mechanism underlying the involvement of non-cyp51A mutations is limited. In this study, using next-generation sequencing, we found that nine independent azole-resistant isolates without cyp51A mutations had normal mitochondrial membrane potential. Among these isolates, a mutation in a mitochondrial ribosome-binding protein, Mba1, conferred multidrug resistance to azoles, terbinafine, and amphotericin B but not caspofungin. Molecular characterization verified that the TIM44 domain of Mba1 was crucial for drug resistance and that the N terminus of Mba1 played a major role in growth. Deletion of mba1 had no effect on Cyp51A expression but decreased the fungal cellular reactive oxygen species (ROS) content, which contributed to mba1-mediated drug resistance. The findings in this study suggest that some non-cyp51A proteins drive drug resistance mechanisms that result from reduced ROS production induced by antifungals.

Candida tropicalis is an emerging medically relevant Candida species. The yeast primarily causes opportunistic infections in intensive care units and is highly prevalent in tropical countries. The genetic diversity within this species is high, and nosocomial transmission has been reported. C. tropicalis genotyping of isolates from low- and middle-income countries is underrepresented when compared with that from high-income countries. Also, in Egypt, only limited genotyping has been conducted for C. tropicalis isolates, while antifungal resistance seems to increase, especially against azoles.
Antifungal susceptibility testing was performed on 64 C. tropicalis isolates from ICU patients collected from multiple hospitals in Alexandria, Egypt. Genotyping by means of short tandem repeat (STR) and whole genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis was performed.
Using antifungal susceptibility testing, fluconazole resistance was observed in 24 isolates (38%), of which 23 harboured an ERG11 G464S substitution, previously shown to cause resistance in Candida albicans. STR genotyping showed that these 23 isolates were related, forming a distinct resistant clade. WGS SNP analysis subsequently confirmed this genetic relationship, although isolates within this clade differed in at least 429 SNPs, suggesting that these were independently introduced.
Overall, STR and WGS SNP analysis of this collection indicates limited C. tropicalis nosocomial transmission in Alexandria, while the presence of this large azole-resistant C. tropicalis clade within this city hampers the treatment of intensive care unit patients.

BACKGROUND: Trichophyton indotineae, a new species of dermatophytes, has become a significant concern in treating dermatophytosis due to the high level of terbinafine resistance reported in India and even worldwide.
OBJECTIVE: This study aimed to report the terbinafine- and itraconazole-resistant T. indotineae in Chinese mainland, by identifying the phylogenetic classification of the isolate strain, and detecting the drug resistance, gene mutation and expression.
METHODS: The skin scales of the patient were cultured on SDA and the isolate was authenticated by DNA sequencing and MALDI-TOF MS. Antifungal susceptibility testing was performed following the M38-A2 CLSI protocol to examine the MICs values of terbinafine, itraconazole, fluconazole, etc. The strain was screened for mutations in the squalene epoxidase (SQLE) gene by Sanger sequencing and detected the expression of CYP51A and CYP51B by qRT-PCR.
RESULTS: A multi-resistant ITS genotype VIII sibling of the T. mentagrophytes complex (T. indotineae) was isolated in Chinese mainland. The strain harbored high terbinafine MIC of > 32 μg/mL and itraconazole MIC of 1.0 μg/mL, which was identified a mutation in the squalene epoxidase gene with amino acid substitution (Phe397Leu, mutation 1191C > A). In addition, overexpression of CYP51A and CYP51B was observed. With multiple relapses, the patient finally achieved clinical cure by itraconazole pulse therapy and topical clotrimazole cream for 5 weeks.
CONCLUSIONS: The first domestic strain of terbinafine- and itraconazole-resistant T. indotineae from a patient in Chinese mainland was isolated. Itraconazole pulse therapy can be an effective method for the treatment of T. indotineae.

UNASSIGNED: The rapid rise of azole resistance in Candida tropicalis causing invasive infections has become a public health concern; however, the prevalence of resistant isolates in urine samples was not well studied, because the clinical significance of candiduria was not unambiguous due to possible host colonization.
UNASSIGNED: We performed a 12-year laboratory-based surveillance study of C. tropicalis causing either invasive infection or candiduria and studied their susceptibility profiles to common antifungal drugs. The complete coding domain sequence of the ERG11 gene was amplified in all fluconazole resistant isolates, and aligned with the wild-type sequence to detect nucleotide mutations.
UNASSIGNED: A total of 519 unique C. tropicalis strains isolates, 69.9% of which were isolated from urine samples and remaining 30.1% were invasive strains. Overall, 16.5% isolates were confirmed to be resistant to fluconazole, of which 91.9% were cross-resistant voriconazole. Of note, at the beginning of surveillance (2010-2011), the fluconazole resistance rates were low in both candiduria and invasive groups (6.8% and 5.9%, respectively). However, the resistant rate in the candiduria group significantly increased to 29.5% since 2012-2013 (p = 0.001) and stayed high since then, whilst the resistance rate in the invasive group only showed a gradually increasing trends till 2021 (p > 0.05). Sequence analysis of ERG11 from fluconazole-resistant strains revealed the prevalence of A395T/W mutations were relatively low (16.7%) in the beginning but reached 87.5-100% after 2014. Moreover, the A395W heterozygous mutation isolates became predominant (>60% of resistant strains) after 2016, and indeed isolates carrying corresponding amino acid substitution (Y132F) was highly resistant to fluconazole with MIC50 exceeded 256 μg/ml.
UNASSIGNED: Our study revealed high azole resistant rate in candiduria with its increasing trends observed much earlier than stains causing invasive infections. Given antimicrobial resistance as a critical \"One Health\" issue, the emergence of antifungal resistance in Candida species that are common commensal colonizers in the human body should be concerned.

Azole resistance is becoming increasingly serious due to the frequent recurrence of fungal infections and the need for long-term clinical prevention. In our previous study, we discovered ORF19.5274 with an unknown function by TMT™ quantitative proteomics technology after fluconazole (FLC) treatment of Candida albicans. In this study, we created the target gene deletion strain using CRISPR-Cas9 editing technology to see if ORF19.5274 regulates azole sensitivity. The data showed that ORF19.5274 was involved in hyphal development and susceptibility to antifungal azoles. Deleting this gene resulted in defective hyphal growth in solid medium, while only a weak lag in the initiation of hyphal development and restoring hyphal growth during the hyphal maintenance phase under liquid conditions. Moreover, intracellular reactive oxygen species (ROS) assay and propidium iodide staining assays showed increased endogenous ROS levels and membrane permeability, but decreased metabolic activity of biofilm in orf19.5274Δ/Δ after treatment with FLC in comparison with either SC5314 or orf19.5274Δ/Δ::ORF19.5274 strains. More importantly, orf19.5274Δ/Δ significantly enhanced the FLC efficacy against C. albicans in infected Galleria mellonella larvae. The above characteristics were fully or partially restored in the complemented strain indicating that the changes caused by ORF19.5274 deletion were specific. In summary, the ORF19.5274 gene is required for hyphal development of C. albicans, and is correlated with the response to antifungal azoles in vitro and in vivo. The identification of ORF19.5275 is promising to expand the potential candidate targets for azoles.