Penicillin and cephalosporin biosyntheses are also regulated by reactive oxygen species.

In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.

Sarocladium and Acremonium infections: New faces of an old opportunistic fungus.

The genera Acremonium and Sarocladium comprise a high diversity of morphologically and genetically related fungi generally found in the environment, although a few species, mainly Sarocladium kiliense and Acremonium egyptiacum, can also be involved in many human infections. Clinical management of opportunistic infections caused by these fungi is very complex, since their correct identification is unreliable, and they generally show poor antifungal response. More than 300 clinical cases involving a broad range of Acremonium/Sarocladium infections have so far been published, and with this review we aim to compile and provide a detailed overview of the current knowledge on Acremonium/Sarocladium human infections in terms of presentation, diagnosis, treatments and prognoses. We also aim to summarise and discuss the data currently available on their antifungal susceptibility, emphasising the promising results obtained with voriconazole as well as their impact in terms of animal infections.

The High Diversity and Variable Susceptibility of Clinically Relevant Acremonium-Like Species in China.

Acremonium-like fungi are emerging as important opportunistic pathogens in cutaneous, subcutaneous and serious invasive infections, especially in immunocompromised and debilitated individuals, and Acremonium infections are usually resistant to antifungal therapy. Several molecular studies have demonstrated that many species in the genus Acremonium are polyphyletic, and currently, the genus is restricted to the family Bionectriaceae (Hypocreales). Molecular identification and in vitro antifungal susceptibility tests of Acremonium-like fungi isolated from human clinical specimens in China were performed in this study. Three genetic loci: the large subunit ribosomal RNA gene (LSU), ribosomal internal transcribed spacer and elongation factor 1-α (EF1-α), were used to assess their taxonomic position for correct identification among various species. The multilocus study of twenty-eight strains showed that these strains were distributed in three main lineages: egyptiacum, Cordycipitaceae and Sarocladium; Acremonium egyptiacum and Sarocladium kiliense were the main species of these strains, and three isolates were too phylogenetically distant to be considered undescribed species. Relatively low minimum inhibitory concentrations (MICs) of 0.25-2 and 0.031-0.5 µg/mL were found for voriconazole and terbinafine for most species, respectively. Varied antifungal activities of ciclopirox olamine, amorolfine and posaconazole were found in our study. However, no antifungal effect of sertaconazole, itraconazole or fluconazole was observed against most strains. This is the first study on Acremonium-like species diversity by multilocus sequence analyses and antifungal susceptibility of clinically relevant isolates in China.

A super-infection in the cornea caused by Stemphylium, Acremonium, and α-Streptococcus.

BACKGROUND: Polymicrobial keratitis with fungus and bacteria can lead to blindness and is challenging to treat. Here, we introduce a case of fungal keratitis caused by two different strains in addition to definite bacterial super-infection caused by an α-Streptococcus sp., and describe the importance of microscopic examination. CASE PRESENTATION: A 74-year-old woman, who had a past history of infection with leprosy, presented with conjunctival hyperaemia, pain, and corneal opacity in her right eye. Under the presumptive diagnosis of infectious keratitis, corneal scrapings were stained by various reagents and inoculated on several agar plates. Microscopic findings of the scrapings revealed fungi and a small number of Gram-positive cocci. Multiple anti-fungal therapies with levofloxacin ophthalmic solution were administered. Although empiric treatment was initially effective, keratitis recurred 10 days after its initiation. Repeated corneal scraping revealed an abundance of Gram-positive chain cocci and a small amount of fungi, resulting in the switching of an antibiotic medication from levofloxacin to moxifloxacin and cefmenoxime. Keratitis resolved gradually after the conversion. Stemphylium sp., Acremonium sp., and α-Streptococcus sp. were simultaneously isolated from the corneal scrapings. CONCLUSIONS: To the best of our knowledge, this is the first case of fungal keratitis caused by Stemphylium sp., and also the first case of super-infection in the cornea caused by two different fungi and one bacterium. Microscopic examination of the corneal scrapings was beneficial in rapid decision of changing to appropriate drug according to the dominancy of pathogenicity.

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds.

The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)D-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 °C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.

Heavy metal ions affecting the removal of polycyclic aromatic hydrocarbons by fungi with heavy-metal resistance.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) is very common in contaminated environments. It is of paramount importance and great challenge to exploit a bioremediation to remove PAHs in these environments with combined pollution. We approached this question by probing the influence of HMs coexisting with PAHs on the removal of PAHs by Acremonium sp. P0997 possessing metal resistance. A removal capability for naphthalene, fluorene, phenanthrene, anthracene, and fluoranthenepresentalone (98.6, 99.3, 89.9, 60.4, and 70 %, respectively) and in a mixture (96.9, 71.8, 67.0, 85.0, and 87.9 %, respectively) was achieved in mineral culture inoculated with Acremonium sp. P0997, and this strain also displayed high resistance to the individual HMs (Mn(2+), Fe(2+), Zn(2+), Cu(2+), Al(3+), and Pb(2+)). The removal of individual PAHs existing in a mixture was differently affected by the separately tested HMs. Cu(2+)enhanced the partition process of anthracene to dead or alive mycelia and the contribution of the biosorption by this strain but imposed a little negative influence on the contribution of biodegradation to the total removal of anthracene individually in a culture. However, Mn(2+) had an inhibitory effect on the partition process of anthracene to dead or alive mycelia and decreased the contributions of both biosorption and biodegradation to the total anthracene removal. This work showcased the value of fungi in bioremediation for the environments with combined pollution, and the findings have major implications for the bioremediation of organic pollutants in metal-organic mixed contaminated sites.

Evaluation of the efficacies of Amphotericin B, Posaconazole, Voriconazole, and Anidulafungin in a murine disseminated infection by the emerging opportunistic Fungus Sarocladium (Acremonium) kiliense.

We evaluated and compared the efficacies of different antifungal drugs against Sarocladium kiliense (formerly Acremonium kiliense), a clinically relevant opportunistic fungus, in a murine model of systemic infection. Three clinical strains of this fungus were tested, and the therapy administered was as follows: posaconazole at 20 mg/kg of body weight (twice daily), voriconazole at 40 mg/kg, anidulafungin at 10 mg/kg, or amphotericin B at 0.8 mg/kg. The efficacy was evaluated by prolonged animal survival, tissue burden reduction, and (1→3)-ß-d-glucan serum levels. In general, the four antifungal drugs showed high MICs and poor in vitro activity. The efficacy of the different treatments was only modest, since survival rates were never higher than 40% and no drug was able to reduce fungal load in all the organs for the three strains tested. Posaconazole, in spite of its high MICs (≥16 µg/ml), showed the highest efficacy. The (1→3)-ß-d-glucan serum levels were equally reduced by all drugs evaluated.

[Microbial models in screening of inhibitors of sterol biosynthesis].

On the base of previously developed microbial models high effective scheme for screening of inhibitors of sterol biosynthesis (ISB) is proposed. It is based on cultivation of halophilic bacteria Halobacterium salinarum (former Halobacterium halobium), possessing mevalonate pathway of sterol biosynthesis, and cultivation of fungus Acremonium fusidioides (former Fusidium coccineum), that is producer of steroid antibiotic fusidin (fusidic acid), which biosynthesis has great similarity (with coincidence of its initial steps till squalene formation) to cholesterol biosynthesis in human organism. In H. salinarum model ISB are revealed as compounds that inhibit test-culture growth, whereas in A. fusidioides test-system they are revealed as compounds that strongly reduce fusidin production without any visible influence on producer's growth. Mevalonate that is one of the crucial intermediates of sterol biosynthesis remove inhibition induced by many microbial metabolites that is the evidence of their action at early stages of sterol biosynthetic pathway, including HMG-CoA reductase step. Both test-systems are developed as micromethod and could be easily mechanized due to miniaturization of microbiological procedures, cultivation in sterile 96-well plates and usage of automatic micropipettes and dispensers. Effectiveness of both test-systems, as well as their sensitiveness, laboriousness and ability to give false-positive or false-negative results in ISB screening work is compared. The proposed scheme of screening of ISB includes microbial models at early steps of screening procedures and Hep G2 test-system at the late step. The preliminary screening of microbial metabolites possessing antifungal activity at initial step is compulsory. Miniaturization and mechanization of microbial processes and purification of producers' culture broth with micro- and ultrafiltration are under consideration as well.

Efficacious treatment of non-dermatophyte mould onychomycosis with topical amphotericin B.

BACKGROUND: Dermatophytes are the main cause of onychomycosis, but various non-dermatophyte moulds (NDMs) are often the infectious agents in abnormal nails. In particular, Fusarium spp. and other NDMs are mostly insensitive to standard onychomycosis treatment with topical agents as well as with oral terbinafine and itraconazole. OBJECTIVE: The aim of this work is to report the efficacy of a topical amphotericin B solution on NDM onychomycosis in a series of 8 patients resistant to multiple conventional topical and systemic treatments. METHODS: Treatment consisted in the application of an optimized amphotericin B solution once daily to the affected nails and surrounding tissue. No mechanical debridement or medications were allowed except for trimming excessively long nails or in some cases occasionally applying urea-based cream to soften thickened nail plates. RESULTS: Onychomycosis was clinically cured in all patients after a 12-month treatment. Mycological cure was obtained in all but 1 patient. CONCLUSIONS: Topical amphotericin B is an efficacious, safe, cheap and easy-to-apply treatment which should be considered as first-line therapy for NDM onychomycosis.

Acremonium species: a review of the etiological agents of emerging hyalohyphomycosis.

Unusual fungal agents that exist environmentally as saprophytes can often lead to opportunistic infections. Hyalohyphomycosis is a group of fungal infections caused by fungi characterized by hyaline septate hyphae and can infect both immunocompetent as well as immunocompromised patients. Many a times it becomes difficult to distinguish a pathogenic and a contaminant fungus, because many such agents can assume clinical significance depending on circumstances. Subcutaneous and invasive fungal infection due to the emerging hyalohyphomycotic fungus, Acremonium, has drawn the attention of clinicians and microbiologists, as a potential pathogen in patients with and without underlying risk factors. Generally considered to be minimally invasive in the past, genus Acremonium has been responsible for eumycotic mycetomas and focal infections in otherwise healthy individuals. It has also been increasingly implicated in systemic fungal diseases. The management with different antifungals in various clinical situations has been very conflicting and hence needs to be carefully evaluated. This overview is an endeavor to consolidate the available clinical infections due to Acremonium and the recommendations on treatment.

Activity profile in vitro of micafungin against Spanish clinical isolates of common and emerging species of yeasts and molds.

A collection of 2,278 isolates belonging to 86 different fungal species was tested with micafungin and eight other drugs using the EUCAST procedures. Micafungin was active against species of Candida and Aspergillus (even azole-resistant species) as well as Penicillium spp., Scedosporium apiospermum, and Acremonium spp. It was inactive for species of Basidiomycota and Mucorales and for multiresistant species such as those of Fusarium.

A case of colonization of a prosthetic mitral valve by Acremonium strictum.

A case of colonization of a prosthetic mitral valve in a 73-year-old Spanish male by the fungus Acremonium strictum W. Gams is described. The valve was replaced due to paravalvular leak and severe insufficiency and the patient died of multiorgan failure. The identity of the fungus was determined by morphological studies and it was confirmed by the analysis of the ITS region sequence analysis. Molecular studies seem to demonstrate that A. strictum is a species complex. The case emphasizes the potential high risk of fungal infection for patients with prosthetic valves.

Electroporation of germinated conidia and young mycelium as an efficient transformation system for Acremonium chrysogenum.

Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.

Pleuritis caused by Acremonium strictum in a patient with colon adenocarcinoma.

Although Acremonium strictum is environmentally widespread as opportunistic mold, it may cause infection in patients who have immunodeficiency problems. In this study, Staphylococcus aureus and A. strictum were isolated from the pleural fluid of a patient with colon adenocarcinoma. The patient did not receive antifungal therapy because the patient died after the isolation of mold. The minimal inhibitory concentrations of amphotericin B, fluconazole, ketoconazole, itraconazole, voriconazole for the A. strictum strain isolated from pleural fluid were 0.125, 256, 2 and 1.5, 0.25 mug ml(-1) respectively. In conclusion, bacteria and fungus, especially opportunistic mold, should be taken into consideration in developing pleuritis in the patients with immune-deficiency.

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10.

AIMS: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid-state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. METHODS AND RESULTS: Solid-state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7.8, but also under 6.4. Using initial pH lower than 6.8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 microg gdm(-1) (g(-1) of dry matter). CONCLUSION: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.

Invasive fungal infection in patients with hematologic disorders in a Brazilian tertiary care hospital.

INTRODUCTION:: Invasive fungal infections (IFIs) are an important complication in immunocompromised individuals, particularly neutropenic patients with hematological malignancies. In this study, we aimed to verify the epidemiology and diagnosis of IFIs in patients with hematologic problems at a tertiary hospital in Goiânia-GO, Brazil. METHODS:: Data from 117 patients, involving 19 cases of IFIs, were collected. The collected data included diagnosis methods, demographics, clinical characteristics, and in vitro susceptibility to different antifungal agents. Among the 19 cases, 12 were classified as proven IFI and 7 as probable invasive aspergillosis with detection of galactomannan in blood and presence of lung infiltrates in radiographic images. Logistic regression analysis showed that the proven and probable IFIs were associated with increased risk of death. Statistical analysis demonstrated that age, sex, and underlying disease were not independently associated with risk of death in IFI patients. RESULTS:: Most bloodstream isolates of Candida spp. exhibited low minimum inhibitory concentrations (MICs) to all antifungal agents tested. Voriconazole and amphotericin had the lowest MICs for Aspergillus spp. and Fusarium spp., but Fusarium spp. showed the least susceptibility to all antifungals tested. Amphotericin B, fluconazole, and itraconazole were found to be inactive in vitro against Acremonium kiliense; but this fungus was sensitive to voriconazole. CONCLUSIONS:: Considering the high number of IFI cases, with crude mortality rate of 6%, we could conclude that IFIs remain a common infection in patients with hematological malignancies and underdiagnosed ante mortem. Thus, IFIs should be monitored closely.

Invasive Acremonium strictum infection in a bone marrow transplant recipient.

We describe an invasive Acremonium strictum infection in a 9-year-old debilitated bone marrow transplant recipient. Outcome was successful, despite resistance to the amphotericin B that was initially administered. A. strictum was isolated from bone and urine cultures. We summarize data on 15 opportunistic invasive infections caused by Acremonium in pediatric hosts reported thus far in the English language literature.

Effects of some benzoxazinoids on in vitro growth of Cephalosporium gramineum and other fungi pathogenic to cereals and on Cephalosporium stripe of winter wheat.

The benzoxazolinones benzoxazolin-2(3H)-one (BOA) and 6-methoxybenzoxazolin-2(3H)-one (MBOA) and selected degradation products of these compounds were examined for their in vitro antifungal activity against Cephalosporium gramineum, Gaeumannomyces graminis var. graminis, and Fusarium culmorum. BOA was also applied to the soil-incorporated inoculum of C. gramineum to test its capability of reducing Cephalosporium stripe disease in winter wheat. MBOA reduced the mycelial growth of G. graminis var. tritici, C. gramineum, and F. culmorum by 50% (EC50) at the concentrations of 77, 134, and 271 microg/mL of corn meal agar, respectively, and the corresponding BOA EC50 values for the fungi were 11, 189, and 456 microg/mL. BOA degradation products 2-amino-3H-phenoxazin-3-one (APO), 2-acetylamino-3H-phenoxazin-3-one (AAPO), and o-aminophenol (o-AP) were much more inhibitory to the growth of C. gramineum and G. graminis var. tritici than the parent compounds. APO, AAPO, and o-AP EC50 values were found to be as low as 0.58, 4.57, and 1.4 microg/mL, respectively, for C. gramineum and 0.78, 2.18, and 0.80 microg/mL for G. graminis var. tritici. These compounds applied at the corresponding concentrations did not significantly affect the mycelial growth of F. culmorum. The treatment of C. gramineum inoculum with a 1% water solution of BOA resulted in a significant reduction infection of winter wheat with C. gramineum as compared to the control with the untreated inoculum,but this treatment was not as effective as the application of a commercial fungicide.