Candida parapsilosis is becoming a predominant non-albicans cause of invasive candidiasis (IC). Echinocandins are the preferred choice for IC treatment and prophylaxis. Resistance to echinocandins in C. parapsilosis has emerged in several countries, but little is known about the susceptibility profile in China or about mechanisms of resistance. Here, we investigated the echinocandin susceptibilities of 2523 C. parapsilosis isolates collected from China and further explored the resistance mechanism among echinocandin-resistant isolates. Anidulafungin exhibited the highest MICs (MIC_50/90, 1 and 2 µg/mL; GM, 0.948 µg/mL), while caspofungin showed better activity (0.5 and 1 µg/mL; 0.498 µg/mL). Significantly higher echinocandin MICs were observed among blood-derived isolates compared to others, especially for caspofungin (GM, 1.348 µg/mL vs 0.478 µg/mL). Isolates from ICU and surgical wards also showed higher MICs. Twenty isolates showed intermediate phenotypes for at least one echinocandin. One was resistant to all three echinocandins, fluconazole and voriconazole, which caused breakthrough IC during long-term exposure to micafungin. WGS revealed this isolate carried a mutation S656P in hotspot1 region of Fks1. Bioinformatics analyses suggested that this mutation might lead to an altered protein conformation. CRISPR Cas9-mediated introduction of this mutation into a reference C. parapsilosis strain increased MICs of all echinocandins 64-fold, with similar results found in the subspecies, C. orthopsilosis and C. metapsilosis. This is the first report of a multi-azole resistant and pan-echinocandin resistant C. parapsilosis isolate, and the identification of a FKS1^S656P conferring pan-echinocandin resistance. Our study underscores the necessity of rigorous management of antifungal use and of monitoring for fungal susceptibility.

We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. Azole susceptibilities of 2318 non-duplicate isolates were determined using CLSI broth microdilution. Isolates were genotyped by a microsatellite typing method. Molecular resistance mechanisms were also studied and functionally validated by CRISPR/Cas9-based genetic alterations. Fluconazole resistance occurred in 2.4% (n = 56) of isolates, and these isolates showed a higher frequency of distribution in ICU inpatients compared with susceptible isolates (48.2%, n = 27/56 versus 27.8%, 613/2208; P = 0.019). Microsatellite-genotyping analysis yielded 29 genotypes among 56 fluconazole-resistant isolates, of which 10 genotypes, including 37 isolates, belonged to clusters, persisting and transmitting in Chinese hospitals for 1–29 months. Clusters harbouring Erg11^Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11^Y132F and Mrr1^Q625K, lent its carriage to being one of the strongest associations with cross-resistance and high MICs of fluconazole (>256 mg/L) and voriconazole (2–8 mg/L), causing transmission across two hospitals. Among mutations tested, Mrr1^Q625K led to the highest-level increase of fluconazole MIC (32-fold), while mutations located within or near the predicted transcription factor domain of Tac1 (D440Y, T492M and L518F) conferred cross-resistance to azoles. This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11^Y132F, and the first demonstration of the mutations Erg11^G307A, Mrr1^Q625K, Tac1^L263S, Tac1^D440Y and Tac1^T492M as conferring resistance to azoles.

Tsukamurella species have been clinically regarded as rare but emerging opportunistic pathogens causing various infections in humans. Tsukamurella pneumonia has often been misdiagnosed as pulmonary tuberculosis due to its clinical presentation resembling tuberculosis-like syndromes. Tsukamurella species have also been confused in the laboratory with other phylogenetic bacteria, such as Gordonia . This study aimed to investigate the clinical, microbiological, and molecular characteristics; species distribution; and antimicrobial susceptibility of Tsukamurella species. Immunodeficiency and chronic pulmonary disease appeared to be risk factors for Tsukamurella pneumonia, and the presence of bronchiectasis and pulmonary nodules on imaging was highly correlated with this infection. The study confirmed that groEL (heat shock protein 60) and secA (the secretion ATPase) genes are reliable for identifying Tsukamurella species. Additionally, the ssrA (stable small RNA) gene showed promise as a tool for discriminating between different Tsukamurella species with the shortest sequence length. In terms of antimicrobial susceptibility, quinolones, trimethoprim/sulfamethoxazole, amikacin, minocycline, linezolid, and tigecycline demonstrated potent in vitro activity against Tsukamurella isolates in our study. The study also proposed a resistance mechanism involving a substitution (S91R) within the quinolone-resistance-determining region of the gyrA gene, which confers resistance to levofloxacin and ciprofloxacin. Furthermore, we found that disk diffusion testing is not suitable for testing the susceptibilities of Tsukamurella isolates to ciprofloxacin, imipenem, and minocycline. In conclusion, our systematic investigation may contribute to a better understanding of this rare pathogen. Tsukamurella species are rare but emerging human pathogens that share remarkable similarities with other mycolic acid–containing genera of the order Actinomycetales, especially Mycobacterium tuberculosis . Consequently, misdiagnosis and therapeutic failures can occur in clinical settings. Despite the significance of accurate identification, antimicrobial susceptibility, and understanding the resistance mechanism of this important genus, our knowledge in these areas remains fragmentary and incomplete. In this study, we aimed to address these gaps by investigating promising identification methods, the antimicrobial susceptibility patterns, and a novel quinolone resistance mechanism in Tsukamurella species, utilizing a collection of clinical isolates. The findings of our study will contribute to improve diagnosis and successful management of infections caused by Tsukamurella species, as well as establishing well-defined performance and interpretive criteria for antimicrobial susceptibility testing.

Objectives: To investigate the epidemiology and molecular typing of 307 clinical Wickerhamomyces anomalus isolates collected in China. Methods: A total of 307 W. anomalus isolates were collected from CHIF-NET, a surveillance network with nationwide coverage, from 2009 to 2021. Antifungal susceptibility of all W. anomalus isolates were tested by broth microdilution according to CLSI methods. Genotyping of all isolates was performed using a panel of polymorphic microsatellite markers. Results: The number of W. anomalus isolates was highest in Northeast China, with 121 strains, accounting for 4.6% of the total Candida spp. isolates in the region. Newborns emerged as the primary community of infection, with 84 isolates (12.7% of all Candida spp. in the newborn group). Most W. anomalus isolates were recovered from blood samples (249 isolates). The isolates were found to exhibit notable ratios of non-wild type to fluconazole and voriconazole (48.5% and 34.5%). A total of 118 microsatellite-based types were identified among a set of 309 isolates that included the 307 clinical isolates along with reference strains ATCC 8168 and CICC 32553. This analysis revealed potential nosocomial outbreaks. Conclusions: Our research is the largest investigation of W. anomalus drug susceptibility thus far, providing foundational data that could contribute to future establishment of resistance breakpoints and enhancing epidemiological surveillance capabilities. Our study suggests the importance of nosocomial surveillance and drug resistance management for W. anomalus.

The infection proportion of Candida orthopsilosis , a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C . orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1–12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.

Fungal infections pose a growing global threat, particularly due to the emergence of multidrug-resistant pathogens. This study reports the first globally documented case of a Wickerhamomyces anomalus isolate (XN272) displaying dual high-level resistance to all tested azoles and echinocandins. Genomic analysis of the strain, which was isolated from a bloodstream infection in a 64-year-old male post-pancreatic surgery, identified two key resistance mechanisms: the azole target gene ERG11 harbored missense mutations (Y140H, K151R) and tandem copy number variations, while the echinocandin target gene FKS1 carried an F665S mutation. Transcriptomic profiling under antifungal exposure suggested an additional resistance mechanism, the upregulation of membrane-associated genes and efflux transporters (e.g., FLU1). Strain XN272 exhibited robust biofilm-forming capacity, a trait linked to reduced drug susceptibility. Despite its resistant phenotype, virulence assessments in immunosuppressed mice showed comparable tissue colonization and clearance rates to strain ATCC 8168. The discovery of pan-azole and pan-echinocandin resistance in W. anomalus highlights the expanding landscape of antifungal resistance and its clinical management challenges.