Emergence of multiple fluconazole-resistant Candida parapsilosis sensu stricto clones with persistence and transmission in China.

OBJECTIVES: We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. METHODS: 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. RESULTS: 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 Erg11Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11Y132F and Mrr1Q625K, 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, Mrr1Q625K 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. CONCLUSIONS: This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11Y132F, and the first demonstration of the mutations Erg11G307A, Mrr1Q625K, Tac1L263S, Tac1D440Y and Tac1T492M as conferring resistance to azoles.

A-to-I RNA Editing in Klebsiella pneumoniae Regulates Quorum Sensing and Affects Cell Growth and Virulence.

Millions of adenosine (A) to inosine (I) RNA editing events are reported and well-studied in eukaryotes; however, many features and functions remain unclear in prokaryotes. By combining PacBio Sequel, Illumina whole-genome sequencing, and RNA Sequencing data of two Klebsiella pneumoniae strains with different virulence, a total of 13 RNA editing events are identified. The RNA editing event of badR is focused, which shows a significant difference in editing levels in the two K. pneumoniae strains and is predicted to be a transcription factor. A hard-coded Cys is mutated on DNA to simulate the effect of complete editing of badR. Transcriptome analysis identifies the cellular quorum sensing (QS) pathway as the most dramatic change, demonstrating the dynamic regulation of RNA editing on badR related to coordinated collective behavior. Indeed, a significant difference in autoinducer 2 activity and cell growth is detected when the cells reach the stationary phase. Additionally, the mutant strain shows significantly lower virulence than the WT strain in the Galleria mellonella infection model. Furthermore, RNA editing regulation of badR is highly conserved across K. pneumoniae strains. Overall, this work provides new insights into posttranscriptional regulation in bacteria.

Study of an antifungal medicine isavuconazole used in treatment of mold infections in China: A plain language summary.

WHAT IS THIS SUMMARY ABOUT?: Molds are types of fungus that can cause sickness and death. Mold infections are increasing in China. Until 2022, medicines that can effectively treat all mold infections were still lacking in China. This summary of a study originally published in the journal Infection and Drug Resistance. The study took place in China and tested a medicine called isavuconazole on mold samples to check if isavuconazole can be used to treat mold infections. Isavuconazole became available in China in January 2022 as a capsule (a hard gel-covered pill filled with a dose of medicine) and in June 2022 as an injection or a shot. WHAT WERE THE RESULTS?: Isavuconazole stopped the growth of most molds. Other medicines were needed at higher amounts to stop the growth of molds. WHAT DO THE RESULTS OF THE STUDY MEAN?: Isavuconazole is another option to treat mold infections in China.

Morphologic identification of clinically encountered moulds using a residual neural network.

The use of morphology to diagnose invasive mould infections in China still faces substantial challenges, which often leads to delayed diagnosis or misdiagnosis. We developed a model called XMVision Fungus AI to identify mould infections by training, testing, and evaluating a ResNet-50 model. Our research achieved the rapid identification of nine common clinical moulds: Aspergillus fumigatus complex, Aspergillus flavus complex, Aspergillus niger complex, Aspergillus terreus complex, Aspergillus nidulans, Aspergillus sydowii/Aspergillus versicolor, Syncephalastrum racemosum, Fusarium spp., and Penicillium spp. In our study, the adaptive image contrast enhancement enabling XMVision Fungus AI as a promising module by effectively improve the identification performance. The overall identification accuracy of XMVision Fungus AI was up to 93.00% (279/300), which was higher than that of human readers. XMVision Fungus AI shows intrinsic advantages in the identification of clinical moulds and can be applied to improve human identification efficiency through training. Moreover, it has great potential for clinical application because of its convenient operation and lower cost. This system will be suitable for primary hospitals in China and developing countries.

In vitro Activity of Isavuconazole and Comparators Against Clinical Isolates of Molds from a Multicenter Study in China.

Purpose: Monitoring antifungal susceptibility patterns for new or established antifungals is imperative. Antifungal resistance is frequent in molds, frequently leading to invasive mold infections (IMIs) in immunocompromised patients with high morbidity and mortality. Limited availability of effective antifungals for treatment of IMIs in China is an enormous challenge. The purpose of this study was to monitor in vitro antifungal resistance profiles of mold isolates from China, with a particular focus on evaluating in vitro isavuconazole (ISA) activity against these isolates, contributing to the treatment guidance in clinics. Methods: We evaluated the in vitro activity of ISA and its comparators (voriconazole [VOR] and amphotericin B [AMB]) against 131 clinical isolates of Aspergillus spp. (n = 105) and Mucorales order (n = 26) collected between 2017 and 2020 from China. Results: ISA and VOR exhibited similar in vitro activity against Aspergillus spp., with minimum inhibitory concentration (MIC)50 of 1 µg/mL and MIC90 of 2 µg/mL, respectively. Overall, AMB was less active than azoles against Aspergillus spp. (MIC50: 4 µg/mL, MIC90: 8 µg/mL). Against the Mucorales order, ISA demonstrated MIC50 of 0.5 µg/mL and MIC90 of 1 µg/mL; however, one strain each of Mucor circinelloides and Syncephalastrum racemosum were resistant to ISA (MICs: >8 µg/mL). VOR exhibited little or no activity (MIC50: 8 µg/mL, MIC90: >8 µg/mL) against the Mucorales order, whereas AMB had MIC50 and MIC90 of 1 µg/mL. Conclusion: This was the first multicenter, in vitro study conducted in China and demonstrated the excellent activities of ISA against most species of the Mucorales order. MIC indicated an advantage over currently available azole antifungals, positioning ISA as a potential alternative to VOR for clinical management of IMIs. As with other antimicrobials, clinicians should employ stewardship and best practices in relation to potential resistance to new azole antifungals.

Sugars promote graft union development in the heterograft of cucumber onto pumpkin.

The use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future.

Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by Aspergillus lentulus in China.

Invasive aspergillosis (IA) due to Aspergillus lentulus is associated with high mortality. In this study, we investigated the clinical and microbiological characteristics of 6 fatal cases of proven or probable IA caused by A. lentulus in China. Underlying immunosuppression, prior antifungal exposure, and intensive care unit (ICU) hospitalization were important risk factors for invasive A. lentulus infection. Phenotypic differences were observed for A. lentulus isolates including slower growth, reduced sporulation, and inability to grow at 48°C, compared with Aspergillus fumigatus complex. ITS sequencing was unable to distinguish A. lentulus from A. fumigatus, but sequencing of the benA, CaM, and rod A loci enabled reliable distinction of these closely related species. Phylogenetic analysis further confirmed that the ITS region had little variation within the Aspergillus section Fumigati while the benA gene offered the highest intraspecific discrimination. Microsatellite typing results revealed that only loci on chromosomes 1, 3, 5, and 6b generated detectable amplicons for identification. All A. lentulus isolates showed in vitro resistance to multiple antifungal drugs including amphotericin B (MIC range 4 to 8 µg/ml), itraconazole (MIC 2 µg/ml), voriconazole (MIC of 4-16 µg/ml), and posaconazole (MIC of 0.5-1 µg/ml). However, MECs for the echinocandin drugs ranged from 0.03-0.25, ≤0.008-0.015, and ≤0.015-0.03 µg/ml for caspofungin, micafungin, and anidulafungin, respectively. A. lentulus is an emerging fungal pathogen in China, causing fatal disease, and clinicians as well as laboratories should be alert to their increasing presence.

Molecular Characterization of Candida parapsilosis by Microsatellite Typing and Emergence of Clonal Antifungal Drug Resistant Strains in a Multicenter Surveillance in China.

Candida parapsilosis is an important species causing invasive candidiasis (IC) in China. The present survey was a national multicenter study of the molecular epidemiology and antifungal susceptibility profiles of C. parapsilosis. Non-duplicate C. parapsilosis isolates were collected from 10 hospitals across China in the CHIF-NET program 2016-2017. Isolates were genotyped using four highly polymorphic microsatellite markers, and susceptibility profiles determined using Sensititre YeastOneTM YO10. A total of 319 C. parapsilosis from separate patients with IC were studied; 49.2, 17.9, and 10.3% isolates were from patients in surgical departments, general intensive care units (ICUs) and neonatal ICUs (NICU), respectively. C. parapsilosis showed good susceptibility to nine antifungal drugs. Microsatellite analysis identified 122 microsatellite (MT) types. Most MT types had sporadic distribution. However, we identified 32 clusters across 10 hospitals; seven clusters were caused by seven endemic genotypes involving five or more isolates in hospitals designated as H01, H02, H06, and H10. These clusters mainly affected surgical departments and ICUs, except for genotype MT42 which was seen in 22 patients from NICU (hospital H06). Of 16 fluconazole-resistant isolates, seven from hospital H02 shared the same genotype MT70, and three from hospital H04 were of genotype MT47. For 37 isolates with non-wild type MICs to 5-flucytosine, 29 were from hospital H01 (genotype MT48). Here we present the first nationwide molecular epidemiology study of C. parapsilosis in China, identified several previously unrecognized clusters, which included antifungal drug resistant isolates. These findings provide important data for control of IC in China.

Investigation of Cryptococcus neoformans magnesium transporters reveals important role of vacuolar magnesium transporter in regulating fungal virulence factors.

Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. Recent studies have demonstrated that metals are critical factors for the regulation of fungal virulence in hosts. In this study, we systemically investigated the function of C. neoformans magnesium transporters in controlling the intracellular Mg balance and virulence-associated factors. We identified three Mg transporters in C. neoformans: Mgt1, Mgt2, and Mgt3. While we could not detect a Mg2+ -related growth phenotype in mgt1 and mgt3 knockout strains, a GAL7p-Mgt2 strain showed significant Mg-dependent growth defects in the presence of glucose. Further analysis demonstrated that MGT2 is a homolog of MNR2 in Saccharomyces cerevisiae, which is localized to the vacuolar membrane and participates in intracellular Mg transport. Interestingly, a transcriptome analysis showed that Mgt2 influenced the expression of 19 genes, which were independent of Mg2+ . We showed that melanin synthesis in C. neoformans required Mg2+ and Mgt2, and that capsule production was negatively regulated by Mg2+ and Mgt2. Repressing the expression of MGT2-induced capsule, which resulted in an increased fungal burden in the lungs. Cumulatively, this study sets the stage for further evaluation of the important role of Mg homeostasis in the regulation of melanin and capsule in C. neoformans.

Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis.

Copper homeostasis is important for virulence of the fungus Cryptococcus neoformans, which can cause lethal meningoencephalitis in humans. Cryptococcus cells encounter high copper levels in the lung, where infection is initiated, and low copper levels in the brain. Here we demonstrate that two Cryptococcus copper transporters, Ctr1 and Ctr4, differentially influence fungal survival during pulmonary infection and the onset of meningoencephalitis. Protein Ctr1 is rapidly degraded under the high-copper conditions found in infected lungs, and its loss has no effect in fungal virulence in mice. By contrast, deleting CTR4 results in a hypervirulent phenotype. Overexpressing either Ctr1 or Ctr4 leads to profound reductions in fungal burden in the lung. However, during the onset of meningoencephalitis, expression of the copper transporters is induced and is critical for Cryptococcus virulence. Our work demonstrates that the fungal cells switch between copper detoxification and acquisition to address different copper stresses in the host.

Iron and copper as virulence modulators in human fungal pathogens.

Fungal pathogens have evolved sophisticated machinery to precisely balance the fine line between acquiring essential metals and defending against metal toxicity. Iron and copper are essential metals for many processes in both fungal pathogens and their mammalian hosts, but reduce viability when present in excess. However, during infection, the host uses these two metals differently. Fe has a long-standing history of influencing virulence in pathogenic fungi, mostly in regards to Fe acquisition. Numerous studies demonstrate the requirement of the Fe acquisition pathway of Candida, Cryptococcus and Aspergillus for successful systemic infection. Fe is not free in the host, but is associated with Fe-binding proteins, leading fungi to develop mechanisms to interact with and to acquire Fe from these Fe-bound proteins. Cu is also essential for cell growth and development. Essential Cu-binding proteins include Fe transporters, superoxide dismutase (SOD) and cytochrome c oxidase. Although Cu acquisition plays critical roles in fungal survival in the host, recent work has revealed that Cu detoxification is extremely important. Here, we review fungal responses to altered metal conditions presented by the host, contrast the roles of Fe and Cu during infection, and outline the critical roles of fungal metal homeostasis machinery at the host-pathogen axis.