Section Fumigati Research Articles

Poultry farms as a One health priority for Aspergillus section Fumigati surveillance

Abstract In poultry production environments, particulate matter (PM) is considered one of the harmful air pollutants [2]. Considering potential biological pathogens, the genus Aspergillus in particular, Aspergillus fumigatus, is a widely recognized human and animal pathogen [3]. Hot-humid farm environments could contribute to the proliferation of these fungi [4]. Therefore, poultry production workers, besides animals [4], may be at a higher risk of inhaling fungi spores [4,5]. This study aimed to determine particulate matter (PM) contamination and analyze the prevalence of Aspergillus section Fumigati in air samples from poultry pavilions. Indoor and outdoor air samples were taken in the 1st, 2nd and 3rd weeks of birds growth cycle (n = 58). Air was collected through the impaction method (MAS-100) onto polycarbonate filters, set at 100 L/min, for 5 min. Followed by DNA extraction, and real-time PCR detection of Aspergillus section Fumigati. For PM evaluation, portable direct-reading equipment (Lighthouse, model 3016 IAQ) was used. Particle concentrations were measured (n = 67) at five distinct fractions during 5 min. Considering particles fractions, PM10 and PM5 where prevalent in 1st (58% PM10; 28% PM5), 2nd (63% PM10; 26% PM5) and 3rd (64% PM10; 25% PM5) weeks. These results evidence that coarse particles (between 2.5 and 10 µm) are the most relevant PM present in this setting. Regarding fungal detection, Aspergillus section Fumigati was widespread inside poultry pavilions (62%, 26 out of 42), highlighting this environment has a potential reservoir of microbial pathogens [7]. Furthermore, PM can act as a host of biological fragments [6]. Overall, in addition to molecular analysis, future studies should consider using culture-based methods to assess the viability of pathogenic microorganisms and their infection potential [3]. From a One Health perspective, quantitative and qualitative research is required to fully understand this environment. Key messages • This study evidence that coarse particles (between 2.5 and 10 µm) are the most relevant PM present and Aspergillus section Fumigati is widespread in poultry pavilions. • A One health intervention should be considered in future studies.

Emerging Aspergillus lentulus infections in Taiwan: clinical and environmental surveillance.

This study aimed to investigate the prevalence and characteristics of Aspergillus lentulus clinical and environmental isolates in Taiwan. Aspergillus isolates obtained from patients at three hospitals and from 530 soil samples across Taiwan were screened. A. lentulus, confirmed by calmodulin sequencing, was subjected to antifungal susceptibility testing and cyp51A analyses. Soil samples yielding A. lentulus were analysed for residues of 25 azole fungicides. Nine A. lentulus isolates were identified, which included seven (1.2%, 7/601) isolates from three antifungal-naïve patients out of 601 Aspergillus section Fumigati clinical isolates and two (0.3%, 2/659) isolates out of 659 Aspergillus soil isolates. All isolates developed white colonies and failed to grow at 48°C. They were susceptible to anidulafungin but showed reduced susceptibility to amphotericin B (AmB), voriconazole and azole fungicides. One heart transplant recipient with proven invasive pulmonary aspergillosis (IPA) initially showed suboptimal response to voriconazole monotherapy but was cured with a combination of voriconazole-caspofungin, liposomal AmB (LAmB)-caspofungin, along with surgery, followed by voriconazole maintenance therapy. Among two critically ill patients with probable IPA, one survived with micafungin, while the other died of aspergillosis despite sequential isavuconazole and LAmB monotherapy. Clinical and environmental isolates sharing identical Cyp51A sequence are identified, matching the Cyp51A sequence of A. lentulus NIID0096. Flusilazole (0.0009 mg/kg) was detected in one soil sample. This study raises concerns about health threat posed by human pathogenic A. lentulus originating from natural environments and underscores the need for increased clinical and laboratory vigilance regarding A. lentulus infections.

Evaluation of Primers OPF-01, P54, and 1253 to Identify A. fumigatus, A. flavus, and A. niger from Polymorphic Patterns Obtained by RAPD-PCR.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

Microbial Assessment in A Rare Norwegian Book Collection: A One Health Approach to Cultural Heritage.

Microbial contamination poses a threat to both the preservation of library and archival collections and the health of staff and users. This study investigated the microbial communities and potential health risks associated with the UNESCO-classified Norwegian Sea Trade Archive (NST Archive) collection exhibiting visible microbial colonization and staff health concerns. Dust samples from book surfaces and the storage environment were analysed using culturing methods, qPCR, Next Generation Sequencing, and mycotoxin, cytotoxicity, and azole resistance assays. Penicillium sp., Aspergillus sp., and Cladosporium sp. were the most common fungi identified, with some potentially toxic species like Stachybotrys sp., Toxicladosporium sp., and Aspergillus section Fumigati. Fungal resistance to azoles was not detected. Only one mycotoxin, sterigmatocystin, was found in a heavily contaminated book. Dust extracts from books exhibited moderate to high cytotoxicity on human lung cells, suggesting a potential respiratory risk. The collection had higher contamination levels compared to the storage environment, likely due to improved storage conditions. Even though overall low contamination levels were obtained, these might be underestimated due to the presence of salt (from cod preservation) that could have interfered with the analyses. This study underlines the importance of monitoring microbial communities and implementing proper storage measures to safeguard cultural heritage and staff well-being.

Risk of fungal exposure in the homes of patients with hematologic malignancies

BackgroundPatients with hematological malignancies are at a high risk of developing invasive fungal infections (IFI) because they undergo several cycles of treatment leading to episodes of neutropenia. In addition, they alternate between hospital stays and periods spent at home. Thus, when an IFI is diagnosed during their hospital stays, it is highly challenging to identify the origin of the fungal contamination. The objective of this study was to analyze at home fungal exposure of 20 patients with leukemia by taking air and water samples in their living residence. MethodsAir was sampled in 3 rooms of each home with a portable air system impactor. Tap water was collected at 3 water distribution points of each home. For positive samples, fungi were identified by mass spectrometry or on the basis of their morphological features. Results85 % of homes revealed the presence in air of Aspergillus spp. and those belonging to the section Fumigati presented the highest concentrations and the greatest frequency of isolation. Concerning mucorales, Rhizopus spp. and Mucor spp. were isolated in air of 20 % and 5 % of dwellings, respectively. In 4 homes, more than 70 % of the fungal species identified in air were potential opportunists; these were mainly Aspergillus spp. with concentrations greater than 20 cfu/m3. The water samples revealed the presence of Fusarium in 3 dwellings, with concentrations up to 80 cfu/L. Finally, for one patient, fungal species isolated during a period of hospitalization were phenotypically similar to those isolated in samples taken at home. For a second patient, a PCR Mucorale was positive on a sample of bronchoalveolar fluid while air samples taken at his home also revealed also the presence of mucorales. ConclusionThe presence of opportunistic fungal species in the air of all the explored homes suggests the need for strengthened preventive measures in the home of immunocompromised patients. It would be interesting to compare the fungi isolated (from patients and from their environment) by genotyping studies aimed at specifying the correspondence existing between fungal species present in the patients’ homes and those responsible for IFI in the same patients.

Assessment of Portuguese fitness centers: Bridging the knowledge gap on harmful microbial contamination with focus on fungi

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m−2.day−1) and Gram-negative bacteria (VRBA: 1.23 CFU m−2.day−1), while for fungi it was the open space area, with 1.86 × 101 CFU m−2.day−1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples’ contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati, as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination.

Highly effective decontamination in a hospital environment: An easy-to-operate, low-cost prototype.

Healthcare-associated infections (HAI) are illnesses acquired during healthcare and are often the most important adverse event during healthcare. With the aim of increasing the effectiveness of disinfection/decontamination processes in the health service with safe and not promote microbial resistance, we propose the development of portable equipment associated with type C ultraviolet light (UVC). The efficiency of the irradiance emitted by the equipment (at dosages 3.5, 5.0, and 60 mJ/cm2) was determined by the action exerted after exposure against four different bacterial (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) and three different fungi (Candida albicans, C. parapsilosis, and Aspergillus section Fumigati). It was possible to observe that all treatments were capable of inactivating the bacterial species evaluated (p < 0.05), causing the irreversible death of these microorganisms. The most effective elimination of fungal agents was at a dose of 60 mJ/cm2 of UVC radiation, with a decrease in the fungal inoculum varying between 94% and 100% in relation to the control without exposure. Thus, our study showed that the application of the portable prototype with UVC light (254 nm) at a distance of 48 mm, allowed an average irradiance of 3.5 mW/cm2, with doses of 3.5 ≈ 60 mJ/cm2 (from 1 to 60 s of exposure), which can promote the total reduction of the bacteria evaluated and significantly reduce fungal growth. Therefore, this prototype could be used safely and effectively in the hospital environment, considerably reducing contamination and contributing to the reduction of healthcare-associated infection risk.

Assessment of waste workers occupational risk to microbial agents and cytotoxic effects of mixed contaminants present in the air of waste truck cabin and ventilation filters

ABSTRACT Workers in the waste-processing industry are potentially exposed to high concentrations of biological contaminants, leading to respiratory and digestive problems and skin irritations. However, few data on the exposure of waste collection truck (WCT) drivers are available. The goal was to document the microbial risk of the waste collection truck (WCT) workers while in the vehicle cab. Long-period sampling using the truck air filters (CAF) and short time ambient air sampling in the cab were used. The potential release of microbial particles from CAFs was also investigated since it could contribute to the microbial load of the cabin air. A combination of analytical methods also helped assess the complex mixture of the biological agents. Aspergillus sections Fumigati and Flavi, E. coli, Enterobacter spp. and Legionella spp. were detected in the CAF of trucks collecting three types of waste. The highest levels of bacteria and fungi were found in the CAF from organic WCT. The highest endotoxin concentrations in CAF were 300 EU/cm2. Most of the CAF showed cytotoxic effects on both lung cells and hepatocytes. Only one mycotoxin was detected in a CAF. The maximal concentrations in the ambient WCT air varied according to the type of waste collected. The highest proportion (84%) of the air samples without cytotoxic effects on the lungs cells was for the recyclable material WCTs. The results revealed the potential microbial risk to workers from a complex mixture of bio-contaminants in the cabs of vehicles collecting all types of waste. The sustained cytotoxic effect indicates the potential adverse health-related impact of mixed contaminants (biological and non-biological) for the workers. Overall, this study highlights the benefits of using complementary sampling strategy and combined analytical methods for a the assessment of the microbial risk in work environments and the need to implement protective measures for the workers. Implications: Exposure to microbial agents is a well-known occupational hazard in the waste management sector. No previous study had evaluated the cytotoxicity of ambient air and ventilation filters to document worker exposure to a combination of contaminants during waste collection. This research confirms the usefulness of ventilation filters for long-term characterization of exposure to infectious agents, azole-resistant fungi, coliform bacteria and mycotoxin. Overall, this study highlights the importance of using several sampling and analysis methods for a comprehensive assessment of microbial risk in work environments, as well as the need to implement appropriate protective measures for collection workers.

Prevalence of Azole-Resistant Aspergillus Section Fumigati Strains Isolated from Romanian Vineyard Soil Samples.

A total of 265 soil samples from various Romanian vineyards were screened for fungi resistant to azoles. Aspergillus section Fumigati isolates exhibited significant resistance to itraconazole and voriconazole, but no azole-resistant Aspergillus fumigatus strains were detected. Six percent of the samples were positive for Aspergillus section Fumigati strains, all of which were azole-resistant. The strains were mainly Aspergillus udagawae (93.75%) and Aspergillus lentulus (6.25%). The predominant azole-resistant Aspergillus species were Aspergillus section Nigri strains, which were found in 75 soil samples. This study highlights the importance of understanding fungal resistance in vineyard soils for both the agricultural and clinical sectors. The presence of resistant strains may affect vine health and wine production while also constituting a challenge in the selection of effective treatments against severe and potentially fatal fungal infections in humans, stressing the importance of species-specific antifungal resistance knowledge.

Antifungal susceptibility of Aspergillusgenus determined by the Etest® method: eleven years of experience at the Instituto Médico La Floresta. Caracas, Venezuela.

Abstract. This research aimed to determine the susceptibility of Aspergil-lus spp. to four antifungal agents using the Etest® method in several clinical samples (respiratory samples, soft tissue, otic tissue, and ocular tissue, among others) from a private health center in Venezuela. Thirty-three strains were evaluated: 11 Aspergillus section Flavi, eight Aspergillus section Fumigati, six Aspergillus section Nigri, four Aspergillus section Terrei, and four Aspergillus spp. A 0.5 McFarland standard suspension of a 5-day culture of each Aspergillusstrain was prepared on Potato Dextrose agar and then inoculated on Sabouraud agar plates with 2% glucose. Voriconazole (VCZ), amphotericin B (AMB), caspo-fungin (CAS), and posaconazole (PCZ) were tested. Minimal inhibitory concen-trations (MIC) in μg/mL were determined after 24 and 48 hours of incubation at 35 °C and th range (R), geometric mean (GM), MIC50, and MIC90 were calcu-lated. The results for the 33 Aspergillus spp. tested after 24 h were the follow-ing: VCZ (R = 0.031- 16; GM = 0.145; MIC50 = 0.125 and MIC90 = 0.5), AMB (R = 0.031-16; GM = 0.644; MIC50 = 0.5 and MIC90 = 8), CAS (R = 0.031-16; GM = 0.1076; MIC50 = 0.063 and MIC90 = 1), PCZ (R =0.031 - 0.5; GM = 0.0755; MIC50 = 0.063 and MIC90 = 0.25). This investigation allowed assessing the an-tifungal susceptibility profiles of Aspergillus spp. isolated from clinical samples by the Etest® method, which is practical, reproducible and easy to perform in microbiology laboratories.

Surveillance of Amphotericin B and Azole Resistance in Aspergillus Isolated from Patients in a Tertiary Teaching Hospital.

The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives.

Antifungal susceptibility patterns for Aspergillus, Scedosporium and Exophiala isolates recovered from cystic fibrosis patients against amphotericin B, and three triazoles and their impact after long term therapies.

In cystic fibrosis (CF) patients, fungal colonization of the respiratory tract is frequently found. Aspergillus fumigatus, Scedosporium genus and Exophiala dermatitidis are the most commonly isolated molds from the respiratory tract secretions of patients with this genetic disorder. The aim of this 5-year surveillance study was to identify trends in species distribution and susceptibility patterns of 212 mold strains identified as Aspergillus spp., Scedosporium spp. and Exophiala spp., isolated from sputum of 63 CF patients who received long term therapy with itraconazole and/or voriconazole. The Aspergillus spp. isolates were identified as members of the sections Fumigati (n=130), Flavi (n=22), Terrei (n=20), Nigri (n=8), Nidulantes (n=1) and Usti (n=1). Among the 16 species of the genus Scedosporium, 9 were S. apiospermum, 3 S. aurantiacum and 4 S. boydii. Among the 14 Exophiala species, all were molecularly identified as E. dermatitidis. Overall, 94% (15/16) of Scedosporium spp., 50% (7/14) of E. dermatitidis, and 7.7% (14/182) of Aspergillus spp. strains, showed high MIC values (≥ 8 µg/ml) for at least one antifungal. Particularly, 8.9% (19/212) of isolates showed high MIC values to amphotericin B, 11.7% (25/212) itraconazole, 4.2% (9/212) to voriconazole and 3.3% (7/212) to posaconazole. In some cases, such as some A. fumigatus and E. dermatitidis isolates recovered from the same patient, susceptibility to antifungal azoles decreased over time. We show that the use of azoles for a long time in CF patients causes the selection/isolation of mold strains with higher MIC values.

86 Fungi and Mycotoxins Occupational Exposure – Unveiling the Contamination in Different Settings from Portugal

Abstract This study intends to present an overview from the assessments of fungal contamination and mycotoxins performed in three different occupational environments (Firefighters headquarters - FFH; Waste collection trucks – WCT and; Cemeteries - CM). Active sampling (impaction and impingement methods) and several passive sampling methods (swabs, settled dust, settled dust filters, electrostatic dust cloths – EDC, cleaning materials,…) were employed. Azole resistance screening, the molecular detection of Aspergillus sections, as well as mycotoxin analysis were also conducted. In FFH microbial contamination didn´t comply with Portuguese IAQ legal requirements in most of the FFH, the widespread of Aspergillus section Fumigati in all the FFH was observed, as well as a trend of multidrug resistance with focus on Fumigati isolates. Several mycotoxins (fumonisin B2, nivalenol, mycophenolic acid and sterigmatocystin) were detected. Concerning WCT Aspergillus sp. (4.18 %) was one of the most prevalent species. Aspergillus section Fumigati was detected in 5 samples. Mycotoxins were detected in filters (N=1) and in settled dust samples (N=16). The mycotoxin detected in the filter was fumonisin B1 and the most detected in settled dust was mycophenolic acid. In what concerns CM, Aspergillus sp. presented the highest counts in DG18 (18.38%) and it wasn´t observed in azole-supplemented SDA media. Mycophenolic acid was detected in one settled dust sample.Overall, our study reveals that a comprehensive sampling approach and combined analytic methods is an important asset in microbial exposure assessments. Furthermore, in all the three occupational environments mycotoxins were detected unveiling this occupational health threat.

Identification of Green Coffee Contaminated Microfungi of the Genus &lt;i&gt;Aspergillus&lt;/i&gt; on the Basis of Polyphasic Approach

The Aspergillus species are widespread in the environment, able to grow at high temperatures and minimal humidity, including in regions with a hot tropical climate. Some species have the potential to produce toxins. This causes the risk of contamination by fungi of the genus Aspergillus and the mycotoxins (MT) produced by them of plant materials and food products, which is possible at any stage of production, transportation and storage. In the volume of coffee imported to the Russian Federation, 85% is accounted for by raw materials (green coffee), for which the risks of mold damage remain at all stages preceding the roasting stage. It is relevant to study the species composition and toxinogenic properties of Aspergillus spp., which contaminate food raw materials for the production of mass consumption foodstuffs. Contaminated products include coffee, which is one of the basic products of the consumer basket. Reliable data on species identification and toxigenic potential of Aspergillus spp. can be obtained only with an integrated approach based on polyphasic taxonomy. The purpose of this work is to study the species composition of fungi of the genus Aspergillus isolated from green coffee using an integrated approach based on polyphasic taxonomy. The species composition of fungi of the genus Aspergillus from the internal mycoflora of 16 samples of green coffee beans of Arabica and Robusta. The species belonging of the isolated 34 singlspore isolates of Aspergillus spp. was determined by cultural and morphological methods and confirmed by molecular genetic analysis, i.e., RT-PCR with DNA markers of conservative sequences (ITS, CaM, β-tub), studied in vitro profile of produced secondary toxic metabolites. The dominance of species of the Niger section was established (A. niger, 90%, and A. tubingensis, A. carbonarius); then, in decreasing order, the species of section Flavi followed (A. flavus, 100%), sections Circumdati (A. ochraceus, 40% and A. westerdijkiae, 60%). In section Fumigati there was one strain of A. fumigatus. Analysis of the profile of toxic metabolites by HPLC-MS/MS in the multi-detection mode showed the production of mycotoxins by the following species: A. niger – fumonisin B2 and ochratoxin A, A. flavus – aflatoxins B1 and B2 together with sterigmatocystin, A. westerdijkiae – ochratoxin A and penicillic acid, A. ochraceus – penicillic acid. Amounts of produced MT show a high toxinogenic potential of Aspergillus spp. Thus, 20 out of 34 strains produced significant amounts of dangerous, regulated mycotoxins: AFL B1, OTA, FB2. Non-toxinogenic isolates were represented by the species A. niger, A. carbonarius, A. tubingensis, A. flavus, and A. fumigatus. A study of the species composition and toxinogenic properties of green coffee contaminants of the genus Aspergillus using a polyphasic approach was carried out in Russia for the first time.

Aspergillus Species Causing Invasive Fungal Disease in Queensland, Australia

BackgroundAspergillus species are important causes of invasive fungal disease, particularly among those with an impaired immune system. Increasing reports have revealed a rising incidence of antifungal drug resistance among Aspergillus spp., particularly among cryptic species. Understanding local antifungal susceptibility patterns is paramount to delivering optimal clinical care.MethodsAspergillus spp. recovered from clinical specimens between 2000 and 2021 from Pathology Queensland were collected. Aspergillus spp. were identified routinely morphologically, and where there was ambiguity or a lack of sporulation, by sequencing of the internal transcribed spacer (ITS) region. All Aspergillus spp. that underwent antifungal susceptibility testing according to the CLSI M38-A3 method and were recorded and included in the study. Amphotericin B, voriconazole, posaconazole, isavuconazole, micafungin, caspofungin, and anidulafungin were tested. Pathology Queensland services all public healthcare facilities in Queensland, Australia.Results236 Aspergillus spp. were identified from clinical specimens during the study period. The most frequent species identified were Aspergillus section Fumigati (n = 119), Aspergillus section Flavi (n = 35), Aspergillus terreus (n = 32) and Aspergillus niger (n = 29). Overall, MIC50/90 values for voriconazole, posaconazole, itraconazole, and isavuconazole were 0.25/1, 0.25/0.5, 0.25/0.5, and 0.5/2 mg/L respectively. Echinocandins demonstrated low MIC values overall with micafungin and anidulafungin both having an MIC50/90 of 0.015/0.03 mg/L. A total of 15 cryptic species were identified; high triazole MIC values were observed with a voriconazole MIC50/90 of 2/8 mg/L. From 2017 to 2021 we observed an increase in incidence of isolates with high voriconazole MIC values. There was no difference in voriconazole MIC values between Aspergillus spp. acquired in North Queensland when compared to Southeast Queensland, Australia.ConclusionIncreasing reports of antifungal resistance among Aspergillus spp. is concerning and warrants further investigation both locally and worldwide. Active surveillance of both the emergence of different Aspergillus spp. and changes in antifungal susceptibility patterns over time is crucial to informing clinicians and treatment guidelines.

Microbial contamination in grocery stores from Portugal and Spain — The neglected indoor environment to be tackled in the scope of the One Health approach

Microbial contamination in grocery shops (GS) should be evaluated since food commodities are commonly handled by workers and customers increasing the risk of food contamination and disease transmission. The aim of this study was to evaluate the microbial contamination in Portuguese and Spanish GS with a multi-approach protocol using passive (electrostatic dust cloths and surface swabs) sampling methods. The molecular detection of Aspergillus sections, mycotoxin analysis, screening of azole resistance as well as cytotoxicity measurement were conducted to better estimate the potential health risks of exposure and to identify possible relations between the risk factors studied.Fruits/vegetables sampling location was the one identified has being the most contaminated (bacteria and fungi) area in GS from both countries. Aspergillus section Fumigati and Fusarium species were observed in samples from Portuguese groceries with reduced susceptibilities to azoles commonly used in the clinical treatment of fungal infections. Fumonisin B2 was detected in Portuguese GS possible unveiling this emergent threat concerning occupational exposure and food safety. Overall, the results obtained raise concerns regarding human health and food safety and must be surveilled applying a One Health approach.

Composition of Culturable Microorganisms in Dusts Collected from Sport Facilities in Finland during the COVID-19 Pandemic.

Sport facilities represent extreme indoor environments due to intense cleaning and disinfection. The aim of this study was to describe the composition of the cultivated microbiota in dust samples collected in sport facilities during the COVID-19 pandemic. A dust sample is defined as the airborne dust sedimented on 0.02 m2 within 28 d. The results show that the microbial viable counts in samples of airborne dust (n = 9) collected from seven Finnish sport facilities during the pandemic contained a high proportion of pathogenic filamentous fungi and a low proportion of bacteria. The microbial viable counts were between 14 CFU and 189 CFU per dust sample. In seven samples from sport facilities, 20-85% of the microbial viable counts were fungi. Out of 123 fungal colonies, 47 colonies belonged to the potentially pathogenic sections of Aspergillus (Sections Fumigati, Nigri, and Flavi). Representatives of each section were identified as Aspergillus fumigatus, A. flavus, A. niger and A. tubingensis. Six colonies belonged to the genus Paecilomyces. In six samples of dust, a high proportion (50-100%) of the total fungal viable counts consisted of these potentially pathogenic fungi. A total of 70 isolates were considered less likely to be pathogenic, and were identified as Aspergillus section Nidulantes, Chaetomium cochliodes and Penicillium sp. In the rural (n = 2) and urban (n = 7) control dust samples, the microbial viable counts were >2000 CFU and between 44 CFU and 215 CFU, respectively, and consisted mainly of bacteria. The low proportion of bacteria and the high proportion of stress tolerant, potentially pathogenic fungi in the dust samples from sport facilities may reflect the influence of disinfection on microbial communities.

Reactive Azlactone Intermediate Drives Fungal Secondary Metabolite Cross-Pathway Generation.

Pathogenic fungi of Aspergillus section Fumigati are known to produce various secondary metabolites. A reported isolation of a compound with an atypical carbon skeleton called fumimycin from A. fumisynnematus prompted us to examine a related fungus, A. lentulus, for production of similar products. Here we report the isolation of fumimycin and a related new racemic compound we named lentofuranine. Detailed analyses revealed that both compounds were assembled by a nonenzymatic condensation of a polyketide intermediate from the terrein biosynthetic pathway and a highly reactive azlactone intermediate produced by an unrelated nonribosomal peptide synthetase carrying a terminal condensation-like domain. While highly reactive azlactone is commonly used in chemical synthesis, its production by a conventional non-metalloenzyme and employment as a biosynthetic pathway intermediate is unprecedented. The observed unusual carbon skeleton formation is likely due to the reactivity of azlactone. Our finding provides another example of a chemical principle being aptly exploited by a biological system.

A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger.

For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

Caspian Sea Mycosands: The Variety and Abundance of Medically Important Fungi in Beach Sand and Water.

Samples from a total of 67 stations, distributed amongst 32 cities along the Caspian Sea coastline, were collected during the summer of 2021 on sunny days. The samples were collected from each station, including both dry/wet sand and shoreline water. The grown samples were primarily analyzed for the macro/microscopic morphologic features of the fungi. Moreover, identification by PCR-RFLP was performed for yeasts, dermatophytes, and Aspergillus sp. strains. Antifungal susceptibility tests were performed for probable-isolated Aspergillus and Candida sp. A total of 268 samples were collected, from which 181 (67.54%) isolates were recovered. Yeast-like fungi and potential pathogenic black fungi were detected in 12 (6.6%) and 20 (11%) of the sand (dry/wet) samples. Potential pathogenic hyaline fungi were identified in 136 (75.1%) samples, in which Aspergillus sp. was the predominant genus and was detected in 76/136 (47.8%) samples as follows: A. section Flavi n = 44/76 (57.9%), A. section Nigri n = 19/76 (25%), A. section Nidulantes n = 9/76 (11.8%), and A. section Fumigati n = 4/76 (5.3%). The most effective azole antifungal agent was different per section: in A. section Fumigati, PSZ; in Aspergillus section Nigri, ITZ and ISZ; in A. section Flavi, EFZ; and in A. section Nidulantes, ISZ. Candida isolates were susceptible to the antifungals tested.