Cryptococcus Neoformans Var Research Articles

Multi-Locus Sequence Typing-Based Genetic Analysis, Antifungal Resistance, and Clinical Prognosis of Cryptococcus neoformans Infections in HIV-Infected Patients in Northern China.

This study aimed to investigate the molecular epidemiological characteristics and drug sensitivity of Cryptococcus from HIV-infected patients and their relationship with patients' prognosis. Seventy-six strains were collected and identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirmed by internal transcribed spacer sequencing. Multi-locus sequence typing was used for the typing of Cryptococcus, and its antifungal susceptibility was tested using FUNGUS 3. The clinical outcomes of the patients were reviewed at 3-, 6-, 9-, and 12-month follow-ups. All strains were Cryptococcus neoformans var. grubii classified into seven sequence types (STs) dominated by ST5, ST31, and a new ST702 strain. The 6- and 9-month survival rates were highest for patients infected with ST31, ST32, and ST174. The antifungal resistant rates were 13.2%, 2.6%, and 1.4% for fluconazole, amphotericin B, and 5-fluorocytosine. Except itraconazole, the minimum inhibitory concentration (MIC) values and wild type (WT)/non-wild type (NWT) of Cryptococcus for antifungal drugs were not related to the clinical prognosis of HIV-infected patients with cryptococcal infection. ST5 was the main ST type, and the new ST702 type was found in a patient who died in a short period of time. Cryptococcus neoformans var. grubii had a relatively high antifungal drug resistance rate to fluconazole. The WT strain accounted for the highest proportions for 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, and itraconazole. The MIC values of Cryptococcus for first-line antifungal drugs showed no relationship with clinical prognosis, implying that MIC values cannot be used to predict the clinical outcome of these patients.

Differential diagnosis to malignant neoplasia: Nasopharyngeal mass in a cat with nasal cryptococcosis in Germany

A 2-year-old Norwegian Forest cat was presented for evaluation of bilateral purulent nasal discharge and stertorous breathing. A computed tomography (CT) scan of the head revealed an intranasal mass of the left nasal cavity extending behind the tube openings and completely obstructing the nasopharynx. Rhinoscopy confirmed a pinkish, shiny mass. CT scan showed both compartments of the right middle ear filled with abnormal soft tissue attenuating material. There was no change in the bony outline of the middle ear. In the endoscopic examination, after endoscopically assisted tympanocentesis, this material in the accessible dorsolateral compartment proved to be classic polypous tissue in addition to highly viscous glue-like secretions. A secondary otitis media due to a drainage disorder was suspected.Using an endoscopic-interventional approach through the nostril, the nasopharyngeal mass was removed for histopathological examination, in order to restore the nasal airway, and to allow tube drainage. In contrast to cats with classical malignant nasal cavity masses, the cat showed several attachment points of the mass and multiple undulating elevations bilaterally in the nasopharyngeal mucosa.Cytological and histopathological examination identified the mass as a fungal granuloma in the context of a cryptococcus infection only rarely observed in Germany. Molecular genetic analysis confirmed an infection with Cryptococcus neoformans var. grubii.A single intranasal and nasopharyngeal endoscopic debridement resulted in a significant improvement of the clinical signs and a complete healing of the right middle ear (including the tympanic membrane) within 14 days, but not in a complete cure of the disease. The cat was therefore treated with oral itraconazole solution for several weeks.The case report shows that nasal cryptococcosis can also affect cats in Germany. Rhinoscopy reveals a nasopharyngeal mass with multiple attachment points, which is unusual for a neoplasia. In addition to the recommended removal of the mass, oral administration of systemic antimycotics is strongly advised.

Disseminated Cryptococcosis Involving Skin and Lung in an Immunocompromised Patient After Kidney Transplantation: A Case Report

Cryptococcus neoformans can cause detrimental cryptococcosis and other severe complications, especially in immunocompromised populations. Disseminated cryptococcosis in organ transplantation patients is extremely rare and often involves lungs, brains, skin, nails, and other organs. In this paper, we report a rare disseminated cryptococcosis case involving skin and lung that presented with recurrent dark brown abscesses and nodules in an immunocompromised female after kidney transplantation. The K-Set detection result of Cryptococcus capsular polysaccharide in serum is positive. The identification of pus culture by Autof MS 1000 indicates that the patient was infected with Cryptococcus neoformans var. grubii. In addition to treatment using liposomal amphotericin B, fluorocytosine, and fluconazole, local extraction of pus and simultaneous injection of liposomal amphotericin B into skin lesions were performed. This comprehensive treatment method proved effective, as the patient recovered from fever and skin symptoms. The innovative treatment may contribute to helping more disseminated cryptococcosis patients.

Severe lobar pulmonary cryptococcosis with a fatal outcome in a clinically normal Allen's swamp monkey (Allenopithecus nigroviridis)

AbstractA clinically normal, 13.5‐year‐old male Allen's swamp monkey (Allenopithecus nigroviridis) developed cardiac arrest and died shortly after anaesthetic induction for a routine examination. Gross examination of the lung revealed consolidation of the left caudal lobe, and cut surfaces exuded a purulent haemorrhagic discharge. Tissue impressions of the pulmonary tissue were consistent with Cryptococcus sp. Histological examination revealed that the infection was isolated to a single lung lobe, and Cryptococcus neoformans var. neoformans was confirmed by culture. This case report discusses the postmortem findings of a monkey with pulmonary cryptococcosis and the importance of considering routine screening in clinically normal guenons.

Synthesis and Evaluation on the Fungicidal Activity of S-Alkyl Substituted Thioglycolurils

A series of S-alkyl substituted thioglycolurils was prepared through the alkylation of corresponding thioglycolurils with halogenoalkanes and tested for their fungicidal activity against six phytopathogenic fungi from different taxonomic classes: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum, and two pathogenic yeasts: Candida albicans and Cryptococcus neoformans var. grubii. A number of S-alkyl substituted thioglycolurils exhibited high activity against Venturia inaequalis and Rhizoctonia solani (85-100% mycelium growth inhibition), and moderate activity against other phytopathogens. S-Ethyl substituted thioglycolurils possessed a high activity against Candida albicans. Additionally, the hemolytic and cytotoxic properties of promising derivatives were determined using human red blood cells and human embryonic kidney cells, respectively. Two S-ethyl derivatives possessed both low cytotoxicity against normal human cells and high fungicidal activity against Candida albicans.

Cryptococcal Meningitis in a Mexican Neurological Center.

Cryptococcal meningitis, one of the most severe infections affecting the central nervous system, often involves severe neurological sequels and high mortality. A retrospective review was performed, including 76 cases admitted in a 10-year period at a neurological referral center in Mexico City. From 68 isolates, 52 fungal specimens were identified as part of the Cryptococcus neoformans var. neoformans complex, 15 as C. neoformans var gattii complex, and one as Cryptococcus non- neoformans/gattii . Higher cryptococcal meningitis incidence and severity were found in HIV-infected men; other risk factors frequently observed were diabetes mellitus and labor exposure to poultry. The main clinical manifestations were subacute headache, cognitive alterations, and photophobia (exclusively in HIV patients). MRI was highly sensitive for pathologic findings such as meningeal enhancements and cryptococcomas, most of them associated to C. neoformans complex. Eleven patients developed severe brain vasculitis, as observed by transcranial Doppler. Hydrocephalus with intracranial hypertension was the most frequent complication. One-half of the population died, and the rest had neurological sequels, mainly neuropsychiatric manifestations and secondary headaches. These patients developed severe functional limitations in performing daily activities in an independent manner.

In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii.

Cryptococcosis is associated with high rates of morbidity and mortality. The limited number of antifungal agents, their toxicity, and the difficulty of these molecules in crossing the blood-brain barrier have made the exploration of new therapeutic candidates against Cryptococcus neoformans a priority task. To optimize the antimicrobial functionality and improve the physicochemical properties of AMPs, chemical strategies include combinations of peptide fragments into one. This study aimed to evaluate the binding of the minimum activity motif of bovine lactoferricin (LfcinB) and buforin II (BFII) against C. neoformans var. grubii. The antifungal activity against these chimeras was evaluated against (i) the reference strain H99, (ii) three Colombian clinical strains, and (iii) eleven mutant strains, with the aim of evaluating the possible antifungal target. We found high activity against these strains, with a MIC between 6.25 and 12.5 µg/mL. Studies were carried out to evaluate the effect of the combination of fluconazole treatments, finding a synergistic effect. Finally, when fibroblast cells were treated with 12.5 µg/mL of the chimeras, a viability of more than 65% was found. The results obtained in this study identify these chimeras as potential antifungal molecules for future therapeutic applications against cryptococcosis.

2043. In Vitro Activity of Manogepix Against 2,810 Fungal Isolates from the SENTRY Surveillance Program (2020-2021) Stratified by Infection Type

Abstract Background Manogepix is a new antifungal with a novel mechanism of action and potent in vitro activity against Candida (except C. krusei), Aspergillus, and rare mold isolates. The manogepix prodrug fosmanogepix is in Phase 2 development for the treatment of invasive mold infections caused by Aspergillus and rare molds (NCT04240886) and has completed Phase 2 studies for candidemia (NCT03604705) and C. auris (NCT04148287) infections. We evaluated the in vitro activity of manogepix and comparators against 2,810 geographically diverse fungal isolates collected in the SENTRY Surveillance Program during 2020-2021 and stratified them by infection type. Methods Antifungal susceptibility testing of manogepix and comparator agents was conducted according to CLSI M27 (2017) and M38 (2017) guidelines. Fungal surveillance isolates were collected from medical centers located in North America (30 sites; 37.9%), Europe (30 sites; 38.4%), Asia-Pacific (11 sites; 14.7%), and Latin America (6 sites; 9.0%). Infection sites included bloodstream infections (BSI; 48.3%), pneumonia in hospitalized patients (PIHP; 19.9%), skin and skin structure infections (SSSI; 7.8%), urinary tract infections (UTI; 3.1%), intra-abdominal infections (IAI; 1.6%), and other infection types (19.3%). Results Manogepix demonstrated potent in vitro activity against Candida spp. isolates (MIC50/90, 0.004-0.03/0.008-0.06 mg/L) regardless of infection type (Table). Similarly, manogepix was highly active against 534 Aspergillus spp. isolates (MEC50/90, 0.015/0.03 mg/L). Manogepix was active against 53 Cryptococcus neoformans var. grubii (MIC50/90, 0.25/1 mg/L), 19 Fusarium spp. (MEC50/90, 0.015/0.06 mg/L), 12 Lomentospora prolificans (MEC50/90, 0.03/0.06 mg/L), and 17 Scedosporium spp. isolates (MEC50/90, 0.06/0.12 mg/L). Conclusion Manogepix showed potent in vitro activity against Candida spp., Aspergillus spp., C. neoformans var. grubii, and rare molds, including Fusarium spp., L. prolificans, and Scedosporium spp. isolates. Notable activity was also demonstrated by manogepix against C. auris isolates. Further clinical development of fosmanogepix in difficult-to-treat, resistant fungal infections is warranted. Disclosures Michael D. Huband, BS, AbbVie: Grant/Research Support|Melinta: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.

Synthesis of the novel cage amides and imides and evaluation of their antibacterial and antifungal activity

Cage amides and imides bearing bicyclo[2.2.1]- and bicyclo[2.2.2]-subunits were synthesized and evaluated both for antimicrobial activity toward five key ESKAPE pathogenic bacteria: one Gram‐positive bacteria methicillin‐resistant Staphylococcus aureus (ATCC 43300), four Gram‐negative bacteria Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa (ATCC 27853) and for antifungal activity towards pathogenic fungal strains Candida albicans (ATCC 90028) and Cryptococcus neoformans var. Grubii (H99; ATCC 208821). Compound VP-4539 with bicyclo[2.2.2]octene motif demonstrated the highest cytotoxic activity towards C. neoformans, while human keratinocytes of HaCaT line, murine fibroblasts of Balb/c 3T3 line and mitogen-activated lymphocytes of peripheral human blood were found to be tolerant to its action. VP-4539 compound did not intercalate into salmon sperm DNA indicating that its cytotoxicity is not related to intercalation into nucleic acid. Keywords: antifungal, antimicrobial, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octene, cytotoxicity, DNA intercalation, human keratinocytes, lymphocytes, сage compounds

P431 Tip to correct the variation position error in applying long-read high-throughput sequencing technology for fungal identification

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Presently, long-read high-throughput sequencing technology has been started applying in the medical mycology field instead of common Sanger sequencing. With its capability to sequence nucleic acid among the large region compared to Sanger sequencing, this technology can detect and analyze both inter- and intra-species sequence variation in internal transcribed spacer (ITS), a fungal house-keeping region, resulting in high accurate fungal identification. However, to set up the long-read high-throughput sequencing technology for fungal identification is quite challenging because of several factors related to the accuracy including the variation position error. So, in this study, we summarize the tip to correct the variation position error in applying long-read high-throughput sequencing technology for fungal identification that we have learnt in the preliminary laboratory setting using the 8 clinical isolates of yeast: Candida albicans (n = 2), C. tropicalis (n = 1), C. glabrata (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (n = 1), Cryptococcus neoformans var. grubii (n = 1). Based on the recruited strains, we found that self-assembling reference sequence generated from raw data of reading by using an auto-program named Canu causes the size-inflated sequence, a larger size calculated as 22.83 ± 7.56% than it should be, resulting in the shift of variation position. This error can be corrected by the alignment process of the reference sequence with the known sequence, both size and position, prior to doing the raw read alignment. The advantage of this process could correct not only for position shifting caused by the analysis process but also the random error generated from nanopore system. To validate this correction protocol, more samples are needed for further study.

Exploring fungal diversity in Antarctic wildlife: isolation and molecular identification of culturable fungi from penguins and pinnipeds

Aims: To survey the diversity of fungal species that may be cultured from Antarctic penguins and pinnipeds, and to test the in vitro susceptibility to triazole drugs of any medically important Aspergillus isolates. Methods: During an expedition to Argentinean Antarctic research stations at Potter Peninsula (South Shetland Islands) and Primavera Cape (Antarctic Peninsula) in February 2019, samples (n = 212) were collected from fur seals (Arctocephalus gazella), elephant seals (Mirounga leonine), leopard seals (Hydrurga leptonyx), Weddell seals (Leptonychotes weddellii) and crabeater seals (Lobodon carcinophaga) and gentoo penguins (Pygoscelis papua). Oral, nasal and rectal swabs and skin/hair brushings were collected from pinnipeds, and skin/feather brushings, cloacal swabs and moulted feathers from penguins. Samples were cultured on Sabouraud dextrose agar and/or potato dextrose agar plates and fungal isolates identified by morphological criteria followed by PCR amplification and DNA sequencing. Antifungal susceptibility of Aspergillus spp. isolates to triazoles was tested. Results: Fungi from 21 genera were isolated from 121/212 (57.1%) samples obtained from pinnipeds and penguins. Among pinnipeds from Potter Peninsula (fur seals and elephants seals), the most frequent fungal species were Debaryomyces hansenii and Rhodotorula mucilaginosa, isolated from the oral, nasal and/or rectal mucosa, and Antarctomyces psychrotrophicus isolated from the skin/hair of all sampled individuals. Among pinnipeds from Primavera Cape (leopard seals, Weddell seals and crabeater seals), the most frequent fungal species were Naganishia adeliensis and Cryptococcus neoformans var. uniguttulatus, isolated from the nasal/oral mucosa of 4/33 (15.2%) and 5/33 (12.1%) animals, respectively. The most frequently isolated fungal species from gentoo penguins (Potter Peninsula), were Pseudogymnoascus pannorum and A. pyschrotrophicus, which both were isolated from skin/feathers of 7/15 (46.7%) birds, and Thelebolus microsporus, isolated from the cloacal mucosa and skin/feathers of 5/15 (33.3%) and 2/15 (13.3%) birds, respectively. Fungi that are potentially pathogenic to both humans and animals, (Aspergillus fumigatus, Asp. flavus, Asp. versicolor, Candida parapsilosis and Microsporum canis) were isolated from 4/38 (10.5%), 1/38 (2.6%), 2/38 (5.3%), 4/38 (10.5%) and 2/38 (5.3%) sampled pinnipeds, respectively. Only non-azole-resistant isolates of Asp. fumigatus and Asp. flavus were identified. Conclusions: The fungal biodiversity in Antarctic pinnipeds and gentoo penguins was explored using standard mycological culture followed by PCR and DNA sequencing. The frequency of fungal carriage varied among animal species, sample type and location. This study constitutes an epidemiologic approach to monitoring of these marine animals for emerging fungal pathogens.

COMPARISON OF C AND N MINERALIZATION AND METAGENOME ANALYSIS OF RHIZOSPHERE SOILS BELONGING TO DIFFERENT Colchicum L. SPECIES

This study is aimed at determining the characteristics and microbiota of soil upon which some Turkish Colchicum (Colchicaceae) species naturally grows. For this aim the rhizosphere soil samples of Colchicum balansae Planch., Colchicum triphyllum Kunze and Colchicum variegatum L. were analysed in this research. The carbon mineralization rate of C. balansae soil at p<0.05 is significantly different from that of the other two soils. In terms of nitrogen mineralization, significant difference exists between all the three soils (p<0.001). Colchicum variegatum rhizosphere was found to have the highest bacterial diversity. The results revealed that 254 bacterial species were common to the three rhizosphere soils, 35.60% of the bacterial species were unique to C. variegatum soil while 21.89% and 22.67% of the bacterial species were unique to C. balansae and C. triphyllum soil respectively. It was found that C. variegatum and C. balansae collected from areas close to each other had the highest number of common bacterial species, while C. triphyllum from the distant region shared 75 with C. variegatum and 19 with C. balansae. Metagenomics analysis reveals that in the rhizophere of C. variegatum, C. balansae and C. triphyllum, Actinobacteria is dominant at phylum level. Likewise, in C. variegatum soil, Nitrosocosmicus and halophilic Halobacter were found to be the dominant archaea. In the soils of C. triphyllum and C. balansae Saccharomycetales were detected, while Cryptococcus neoformans var. grubii H99 was exclusively detected in C. balansae soil. Significant difference (p<0.05) was observed in C. variegatum rhizosphere soil in terms of organic carbon (C%) and carbon mineralization from the other two soil samples. Significant differences were observed in all three soils in terms of nitrogen content, and the C. triphyllum rhizosphere soil was significantly different from the others in terms of available phosphorus content (p<0.05). This study showed that biological as well as the physico-chemical properties of the rhizosphere soil regulate soil microbial diversity and density and by extension influences their activity which evidently manifests itself in carbon and nitrogen mineralisation.

Phenotypic Diversity of Cryptococcus neoformans var. neoformans Clinical Isolates from Localized and Disseminated Infections.

Cryptococcus neoformans var. neoformans is the second most prevalent agent of cryptococcosis in central Europe. Infections mostly present with localized skin and disseminated infections. Previous studies did not find these presentations to be determined by the fungal genotype as detected by multilocus sequence typing (MLST). However, phenotypic fungal traits may impact clinical presentation. Here, we studied the growth and virulence factors of C. neoformans var. neoformans isolates from disseminated and localized infections and an environmental isolate. We used coincubation with Acanthamoeba castellanii and the Galleria mellonella infection model to identify phenotypic characteristics potentially associated with clinical presentation. Clinical isolates of C. neoformans var. neoformans present a substantial phenotypic variability. Median survival of G. mellonella varied between 6 and 14 days. C. neoformans var. neoformans isolates from disseminated infections showed stronger melanization and larger capsules. They demonstrated superior uptake into an amoeba and increased cytotoxicity for the amoeba. Differences of strains from localized and disseminated infections in coincubation with amoeba are in line with the importance of phagocytes in the pathogenesis of disseminated cryptococcosis. Phenotypic traits and non-vertebrate infection models may help understand the virulence potential of C. neoformans var. neoformans isolates.

Antimicrobial action of arylsulfonamides bearing (aza)norbornane and related motifs: evaluation of new promising anti-MRSA agents

Arylsulfonamides bearing (aza)norbornane and related motifs were evaluated for: (1) antimicrobial activity toward five key ESKAPE pathogenic bacteria, one Gram‐positive bacteria methicillin‐resistant Staphylococcus aureus (MRSA, ATCC 43300), four Gram‐negative bacteria, Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Acinetobacter baumannii (ATCC 19606), and Pseudomonas aeruginosa (ATCC 27853), and (2) antifungal activity towards two pathogenic fungal strains - Candida albicans (ATCC 90028) and Cryptococcus neoformans var. Grubii (H99; ATCC 208821). Four compounds with 4-nitrobenzenesulfonamide motif (VP-4556, VP-4604, VP-4605, and VP-4509) demonstrated high toxicity towards methicillin‐resistant Staphylococcus aureus (ATCC 43300) with the MIC value of 14.7–49.3 µM. These compounds also possessed high antibacterial activity towards gram-negative bacteria P. aeruginosa (ATCC9027) with the MIC value of 460–555 µM. According to the results of toxicity studies of the compounds to HEK-293, HaCaT, Balb/c 3T3, red blood cells and normal mitogen-activated lymphocytes, three compounds - VP-4556, VP-4604 and VP-4509 - bearing azanorbornene, octahydro-3,5-methanocyclopenta[b]pyrrole and bio-isosteric piperidine motifs were selected for further studies as biocompatible agents with promising antimicrobial activity.

Epitope-Based Immunoinformatic Approach on Heat Shock 70 kDa Protein Complex of Cryptococcus neoformans var. grubii.

Introduction Cryptococcosis is a ubiquitous opportunistic fungal disease caused by Cryptococcus neoformans var. grubii. It has high global morbidity and mortality among HIV patients and non-HIV carriers with 99% and 95%, respectively. Furthermore, the increasing prevalence of undesired toxicity profile of antifungal, multidrug-resistant organisms and the scarcity of FDA-authorized vaccines were the hallmark in the present days. This study was undertaken to design a reliable epitope-based peptide vaccine through targeting highly conserved immunodominant heat shock 70 kDa protein of Cryptococcus neoformans var. grubii that covers a considerable digit of the world population through implementing a computational vaccinology approach. Materials and Methods A total of 38 sequences of Cryptococcus neoformans var. grubii's heat shock 70 kDa protein were retrieved from the NCBI protein database. Different prediction tools were used to analyze the aforementioned protein at the Immune Epitope Database (IEDB) to discriminate the most promising T-cell and B-cell epitopes. The proposed T-cell epitopes were subjected to the population coverage analysis tool to compute the global population's coverage. Finally, the T-cell projected epitopes were ranked based on their binding scores and modes using AutoDock Vina software. Results and Discussion. The epitopes (ANYVQASEK, QSEKPKNVNPVI, SEKPKNVNPVI, and EKPKNVNPVI) had shown very strong binding affinity and immunogenic properties to B-cell. (FTQLVAAYL, YVYDTRGKL) and (FFGGKVLNF, FINAQLVDV, and FDYALVQHF) exhibited a very strong binding affinity to MHC-I and MHC-II, respectively, with high population coverage for each, while FYRQGAFEL has shown promising results in terms of its binding profile to MHC-II and MHC-I alleles and good strength of binding when docked with HLA-C∗12:03. In addition, there is massive global population coverage in the three coverage modes. Accordingly, our in silico vaccine is expected to be the future epitope-based peptide vaccine against Cryptococcus neoformans var. grubii that covers a significant figure of the entire world citizens.

Synthetic modifications of abietane diterpene acids to potent antimicrobial agents

Among abietane type semisynthetic diterpenoids, a series of quinopimaric and maleopimaric acid derivatives modified at the carboxyl and carbonyl groups, and in ring E were synthesised to obtain new compounds with antimicrobial potency against Mycobacterium tuberculosis H37Rv and key ESKAPE pathogens. It was found that compound 8 exhibited low toxicity to human embryonic kidney cell line HEK-293 (> 32 μg/mL) and showed significant bacteriostatic activity against methicillin-resistant Staphylococcus aureus (MRSA) (MIC ≤ 0.25 µg/mL) and excellent antifungal activity against Cryptococcus neoformans var. grubii (MICs ≤0.25 µg/mL) being ≈4 and ≈30 fold more active than vancomycin and fluconazole. It also showed moderate activity against fungus Candida albicans (MIC ≤ 0.25 µg/mL). Compound 9 inhibited M. tuberculosis H37Rv with MIC of 1.25 µg/mL. The docking studies suggest possible interactions of the leading compounds with the molecular targets.

In vitro Antifungal Susceptibility Profiles of Cryptococcus neoformans var. grubii and Cryptococcus gattii Clinical Isolates in Guangxi, Southern China.

This study analyzed the in vitro drug sensitivity of Cryptococcus spp. from Guangxi, Southern China. One hundred three strains of Cryptococcus were recovered from 86 patients; 14 were HIV positive and 72 were HIV negative. Ninety-two strains were identified as Cryptococcus neoformans var. grubii, while 11 strains were identified as Cryptococcus gattii (5 C. gattii sensu stricto and 6 Cryptococcus deuterogattii). The recovered strains were tested against commonly used antifungal drugs (fluconazole, amphotericin B, 5-fluorocytosine, itraconazole, and voriconazole) and to novel antifungal drugs (posaconazole and isavuconazole) using CLSI M27-A4 method. The results showed that all isolates were susceptible to most antifungal drugs, of which the minimum inhibitory concentration (MIC) ranges were as follows: 0.05–4 μg/ml for fluconazole, 0.25–1 μg/ml for amphotericin B; 0.0625–2 μg/ml for 5-fluorocytosine, 0.0625–0.25 μg/ml for itraconazole, 0.0078–0.25 μg/ml for voriconazole, 0.0313–0.5 μg/ml for posaconazole, 0.0020–0.125 μg/ml for isavuconazole for C. neoformans var. grubii isolates, and 1–16 μg/ml for fluconazole, 0.125–1 μg/ml for 5-fluorocytosine, 0.25–1 μg/ml for amphotericin B, 0.0625–0.25 μg/ml for itraconazole, 0.0156–0.125 μg/ml for voriconazole, 0.0156–0.25 μg/ml for posaconazole, and 0.0078–0.125 μg/ml for isavuconazole for C. gattii isolates. Furthermore, some C. neoformans var. grubii isolates were found to be susceptible-dose dependent to 5-fluorocytosine and itraconazole. In addition, a reduction in the potency of fluconazole against C. gattii is possible. We observed no statistical differences in susceptibility of C. neoformans var. grubii and C. gattii in the tested strains. Continuous observation of antifungal susceptibility of Cryptococcus isolates is recommended to monitor the emergence of resistant strains.

Antifungal susceptibility testing with YeastONE™ is not predictive of clinical outcomes of Cryptococcus neoformans var. grubii fungemia.

Mortality rates due to Cryptococcus neoformans var. grubii fungemia remain significant despite treatment with antifungal drugs. The predictive function of antifungal susceptibility and its correlation with treatment outcome remains controversial. A retrospective study was conducted from January 1, 2009, to December 31, 2016, on 85 patients with C. neoformans var. grubii fungemia confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Antifungal drug susceptibility was determined using the YeastONE™ colorimetric broth microdilution method coupled with Vizion™ System following the Clinical and Laboratory Standards Institute guidelines. Six antifungal agents-amphotericin B, fluconazole, flucytosine, itraconazole, posaconazole, and voriconazole-were tested. The patients' demographic data and clinical information were abstracted for further analyses. Antifungal regimens consisting of amphotericin B with or without fluconazole or flucytosine were administered for induction treatment of these patients, followed with intravenous or oral fluconazole for maintenance therapy. Clinical outcomes were defined by 14- and 30-day mortality rates. Risk factors associated with outcomes were fitted in a logistic regression model by univariate or multivariate method. Eighty-five patients with C. neoformans var. grubii fungemia were enrolled in the study. The Sequential Organ Failure Assessment Score, Glasgow Coma Scale, Charlson comorbidity score, and adequate duration of therapy for amphotericin B were predictors for mortality in univariate analysis. Antifungal susceptibility testing with YeastONE™ does not predict clinical outcomes of C. neoformans var. grubii fungemia. Greater disease severity, high comorbidities, poor consciousness level, and inappropriate treatment were associated with increased mortality in cryptococcemia cases.

Novel Phenyl‐Based Bis‐quaternary Ammonium Compounds as Broad‐Spectrum Biocides

Herein we report the synthesis and microbiological evaluation of novel phenyl based bis-quaternary ammonium compounds (bis-QACs). Using a simple 2-step synthetic route from dibromo- and dihydroxybenzenes, we obtained a structurally diverse broad panel of bis-QACs with topologically distinct bridging connections between pyridinium heads. Selected analogs possessed potent broad-spectrum biocidal activity against both bacterial and fungal pathogens: methicillin-resistant Staphylococcus aureus (ATCC 43300); Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (ATCC 90028), Cryptococcus neoformans var. grubii (ATCC 208821). Promising compounds displayed minimum inhibitory concentrations (MIC) values ≤0.25 μg/mL alongside improved cytotoxicity and hemolytic profiles compared to modern antiseptics. Thus, synthesized bis-QACs represent a promising class of biocides with the potential to replace existing household sanitizers.

Primary discovery of 1-aryl-5-substituted-1H-1,2,3-triazole-4-carboxamides as promising antimicrobial agents

Three series of novel 1H-1,2,3-triazole-4-carboxamides: 1-aryl-5-alkyl/aryl-1H-1,2,3-triazole-4-carboxamides, 1-aryl-5-amino-1H-1,2,3-triazole-4-carboxamides and 1,2,3-triazolo[1,5-a]quinazoline-3-carboxamides were synthesized via base-mediated click azide reactions. Compounds were evaluated for their antimicrobial activities against primary pathogens: Gram-positive and Gram-negative bacterial strains Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, as well as fungal strain Cryptococcus neoformans var. grubii and Candida albicans. Compounds exhibiting moderate to good activities were selected for SAR analysis. Several 5-methyl-1H-1,2,3-triazole-4-carboxamides 4d, 4l, 4r, showed potent antibacterial effect against S. aureus. On the contrary, 5-amino-1H-1,2,3-triazole-4-carboxamide 8b and [1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide 9a were active against pathogenic yeast C. albicans. Thus, compound 4l under 1 µM demonstrated 50% growth inhibition against S. aureus. At the same concentration, the compound 9a killed approx. 40% of C. albicans cells. In general, these compounds demonstrated selective action and no significant impact on the viability of human keratinocytes of HaCaT line.