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Biofilm-associated polymicrobial infections tend to be challenging to treat. Candida albicans and Staphylococcus aureus are leading pathogens due to their ability to form biofilms on medical devices. However, the therapeutic implications of their interactions in a host is largely unexplored. In this study, we used a mouse subcutaneous catheter model for in vivo-grown polymicrobial biofilms to validate our in vitro findings on C. albicans-mediated enhanced S. aureus tolerance to vancomycin in vivo. Comparative assessment of S. aureus recovery from catheters with single- or mixed-species infection demonstrated failure of vancomycin against S. aureus in mice with co-infected catheters. To provide some mechanistic insights, RNA-seq analysis was performed on catheter biofilms to delineate transcriptional modulations during polymicrobial infections. C. albicans induced the activation of the S. aureus biofilm formation network via down-regulation of the lrg operon, repressor of autolysis, and up-regulation of the ica operon and production of polysaccharide intercellular adhesin (PIA), indicating an increase in eDNA production, and extracellular polysaccharide matrix, respectively. Interestingly, virulence factors important for disseminated infections, and superantigen-like proteins were down-regulated during mixed-species infection, whereas capsular polysaccharide genes were up-regulated, signifying a strategy favoring survival, persistence and host immune evasion. In vitro follow-up experiments using DNA enzymatic digestion, lrg operon mutant strains, and confocal scanning microscopy confirmed the role of C. albicans-mediated enhanced eDNA production in mixed-biofilms on S. aureus tolerance to vancomycin. Combined, these findings provide mechanistic insights into the therapeutic implications of interspecies interactions, underscoring the need for novel strategies to overcome limitations of current therapies. ispartof: VIRULENCE vol:12 issue:1 pages:835-851 ispartof: location:United States status: published

Candida glabrata is the second leading cause of candidemia in many countries and is one of the most concerning yeast species of nosocomial importance due to its increasing rate of antifungal drug resistance and emerging multidrug-resistant isolates. Application of multilocus sequence typing (MLST) to clinical C. glabrata isolates revealed an association of certain sequence types (STs) with drug resistance and mortality. The current C. glabrata MLST scheme is based on single nucleotide polymorphisms (SNPs) at six loci and is therefore relatively laborious and costly. Furthermore, only a few high-quality C. glabrata reference genomes are available, limiting rapid analysis of clinical isolates by whole genome sequencing. In this study we provide long-read based assemblies for seven additional clinical strains belonging to three different STs and use this information to simplify the C. glabrata MLST scheme. Specifically, a comparison of these genomes identified highly polymorphic loci (HPL) defined by frequent insertions and deletions (indels), two of which proved to be highly resolutive for ST. When challenged with 53 additional isolates, a combination of TRP1 (a component of the current MLST scheme) with either of the two HPL fully recapitulated ST identification. Therefore, our comparative genomic analysis identified a new typing approach combining SNPs and indels and based on only two loci, thus significantly simplifying ST identification in C. glabrata. Because typing tools are instrumental in addressing numerous clinical and biological questions, our new MLST scheme can be used for high throughput typing of C. glabrata in clinical and research settings. We thank Dibyendu Kumar (Rutgers University) for help with C. glabrata PacBio sequencing. This work was supported by NIH 5R01AI109025 to D.S.P. TG group acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the Catalan Research Agency (AGAUR) SGR423; from the European Union's Horizon 2020 research and innovation programme (ERC-2016-724173); from the Gordon and Betty Moore Foundation (Grant GBMF9742) and from the Instituto de Salud Carlos III (INB Grant PT17/0009/0023 – ISCIII-SGEFI/ERDF).

Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC50 of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC50 in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections.

Candida meningitis in neurosurgical patients is relatively unusual but is associated with a high mortality rate. We present our experience with this infection and discuss the clinical characteristics, treatment options and outcomes. We retrospectively reviewed neurosurgical patients with multiple positive cerebrospinal fluid (CSF) culture results in our hospital from January 2013 to December 2019. Nine patients were available for review according to our inclusion and exclusion criteria. Four species of Candida were isolated from the CSF samples and Candida albicans accounted for half of all infections. Eight infections were associated with ventricle peritoneal shunt, lumbar cistern peritoneal shunt or external ventricular drain. All of these foreign intracranial materials were removed or changed and all the patients received antifungal treatment, including fluconazole and/or voriconazole. It is associated with severe long-term outcomes in survivors and a mortality rate that reaches 11.1%. Prior treatments with broad-spectrum and high-grade antibiotics and anaemia are possible risk factors for Candida meningitis. We advise that foreign intracranial material should be removed or changed as early as possible and the timing of re-shunt operation can be 1 month after control of Candida meningitis has been achieved, with several negative CSF culture results.

It is known that cetylpyridinium chloride (CPC) has in vitro and in vivo antifungal action against Candida albicans, with advantages over other common antiseptics. A CPC delivery-controlled system, transported in polymer nanofibers (PVP/PMMA), was developed to increase the bioavailability of the drug in contact with the oral mucosa. The objectives of this study were to determine if CPC in nanofiber has antifungal action against C. albicans, and in what concentration it must be incorporated, so that the fraction released can yield an inhibitory concentration. The nanofiber was prepared by electrospinning, and sterilized with gamma irradiation. Nanofiber disks with 0.05%, 1.25%, 2.5% and 5% CPC, with 5% miconazole (MCZ) and with no drug, as well as filter paper disks with 5% CPC, with 5% MCZ and with no drug were used in this study. A Candida albicans suspension (ATCC 90028) was inoculated in Mueller-Hinton Agar plates. The disks were placed on the plates and the inhibition zone diameters were measured 48h later. The nanopolymeric disks contracted in contact with the agar. All the concentrations of CPC incorporated in the nanofiber presented inhibitory action against C. albicans. Concentrations of 2.5% and 5% CPC presented a significant advantage over the nanofiber with no drug, proving the antifungal action of CPC. Under these experimental conditions, 5% CPC has greater inhibitory action against C. albicans than 5% MCZ, both in nanofiber and in filter paper. A modification made in the polymer to decrease the contraction rate may allow a larger inhibition zone to be maintained, thereby increasing the clinical usefulness of the polymer.

The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive P(PGK1) promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00056-8) contains supplementary material, which is available to authorized users.

The treatment of invasive candidiasis has changed greatly in the past decade and must continue to evolve if we are to improve outcomes in this serious infection. A review of recent history may provide insights for the future. The morbidity and mortality of invasive candidiasis remain difficult to measure despite an obvious clinical burden. Current treatment guidelines now recommend echinocandins as first-line empirical treatment, with fluconazole as an acceptable alternative for selected patients, reflecting the efficacy demonstrated by echinocandins and increasing resistance observed with fluconazole. The selection of antifungal therapy now must consider not only resistance but also the shift in predominance from Candida albicans to non-albicans species, notably Candida glabrata. The recent emergence of Candida auris has been met with great interest, although the longer-term implications of this phenomenon remain unclear. The broad goal of treatment continues to be administration of safe, efficacious antifungal therapy as soon as possible. Diagnostic methods beyond traditional blood culture present an opportunity to shorten the time to an accurate diagnosis, and earlier treatment initiation based on prophylactic and empirical or pre-emptive strategies seeks to ensure timely therapeutic intervention. In addition, there are novel agents in the antifungal pipeline. These developments, as well as ongoing studies of dosing, toxicity and resistance development, are important items on the current research agenda and may play a role in future changes to the treatment of invasive candidiasis.

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. It can cause both superficial and serious systemic disease. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. The aim of this study was to investigate the effect of sodium diclofenac and aspirin on germs tube formation of different Candida albicans strains. Prostaglandins may play an important role in fungal colonization. Nonsteroidal anti-inflammatory drugs are inhibitors of the cyclooxygenase isoenzymes. These drugs specifically block the biosynthesis of mammalian prostaglandins by inhibiting one or both of cyclooxygenase isoenzymes. In tests for germ tube formation sodium diclofenac reduced the filamentation to the 12.5%- 5.1%. In the presence of aspirin the filamentation was reduced up to 85-45% depending on the tested strain. Our results suggest that cyclooxygenase-depending synthesis of fungal prostaglandins is important for morphogenesis and fungal virulence. Inhibitors of cyclooxygenase isoensymes (aspirin and diclofenac) are effective in decreasing germ tube formation of Candida albicans.

Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals., Research leading to these results received partial funding from the European Community's Seventh Framework Program (FP7, 2007-2013), Research Infrastructures Action, under grant agreement No. FP7-228310 (EMbaRC project). Thanks are also due to Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil) for funding support

Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods.

The oral microbiota of humans and animals is made up of a wide variety of yeasts and bacteria, but microbiota of dogs is not totally described. Although such identification is an important step to establish the etiopathogenesis and adequate therapy for the periodontal disease The aim of this study was to evaluate and correlate oral alterations with the presence of yeasts in oral cavity of female dogs. After clinical evaluation samples from healthy and from dogs with oral diseases were obtained from three different oral sites by swabs, curettes, millimeter periodontal probes and HA membrane tip in cellulose ester. Yeast identification was performed through macroscopic and microscopic colony features and biochemical tests. Dental calculus was the most prevalent occurrence in the oral cavity of 59 females. However, the isolation of yeasts was significantly higher (p < 0.05) in animals suffering from halitosis. Eleven yeast species were identified, namely: Malassezia pachydermatis, Rhodotorula spp., Candida albicans, C. catenulata, C. famata, C. guilliermondii, C. parapsilosis, C. intermedia, Trichosporon asahii, T. mucoides and Cryptococcus albidus. It could be concluded that the yeasts are part of the microbiota from the different sites of the oral cavity of the female canines studied without causing any significant alterations except halitosis.

Removal of pathogenic organisms from reprocessed surgical instruments is essential to prevent iatrogenic infections. Some bacteria can make persistent biofilms on medical devices. Contamination of non-disposable equipment with prions also represents a serious risk to surgical patients. Efficient disinfection of prions from endoscopes and other instruments such as high-resolution cameras remains problematic because these instruments do not tolerate aggressive chemical or heat treatments. Herein, we develop a new washing system that uses both the alkaline and acidic water produced by electrolysis. Electrolyzed acidic water, containing HCl and HOCl as active substances, has been reported to be an effective disinfectant. A 0.15% NaCl solution was electrolyzed and used immediately to wash bio-contaminated stainless steel model systems with alkaline water (pH 11.9) with sonication, and then with acidic water (pH 2.7) without sonication. Two bacterial species (Staphylococcus aureus and Pseudomonas aeruginosa) and a fungus (Candida albicans) were effectively removed or inactivated by the washing process. In addition, this process effectively removed or inactivated prions from the stainless steel surfaces. This washing system will be potentially useful for the disinfection of clinical devices such as neuroendoscopes because electrolyzed water is gentle to both patients and equipment and is environmentally sound.

Objective: Candida infections have increased due to transplant patients, prolonged ICU stay and invasive procedures. The most common isolated strain is C. albicans. The aim of this study was to evaluate the distribution of Candida isolates at Tepecik Education and Research Hospital. Materials and Methods: Yeast like fungi were isolated between 13.01.2010 and 19.08.2011 at Mycology Laboratory. The identification was done by conventional methods and carbohydrate assimilation profile using the ID32C identification system (Biomerieux, France). Results: Yeast like fungi were isolated from 337 clinical specimens. They consisted of urine, blood culture, respiratory specimen and wound. The most isolated yeast strains were C.albicans (38.6%), C.tropicalis (13.9%), C. parapsilosis (28.4%), C.glabrata (7.4%), C.krusei (3.8%). Conclusion: Recently there is an increment in Candida infections. In this study the most common strain was C.albicans and the rate C. glabrata and C. krusei isolates were lower than expected. C. parapsilosis was the most isolated strain in blood cultures and this may be due to invasive procedures and the use of indwelling catheters.

Candida dubliniensis is closely related to Candida albicans, a major causative agent of candidiasis, and is primarily associated with oral colonization and infection in human immunodeficiency virus (HIV)-positive patients. Despite the high similarity of genomic and phenotypic features between the 2 species, C. dubliniensis is much less virulent and less prevalent than C. albicans. The ability to change morphological phenotypes is a striking feature of Candida species and is linked to virulence. In this study, we report a novel phenotype, the gray phenotype, in C. dubliniensis. Together with the previously reported white and opaque cell types, the gray phenotype forms a tristable phenotypic switching system in C. dubliniensis that is similar to the white-gray-opaque tristable switching system in C. albicans. Gray cells of C. dubliniensis are similar to their counterparts in C. albicans in terms of several biological aspects including cellular morphology, mating competence, and genetic regulatory mechanisms. However, the gray phenotypes of the 2 species have some distinguishing features. For example, the secreted aspartyl protease (Sap) activity is induced by bovine serum albumin (BSA) in gray cells of C. albicans, but not in gray cells of C. dubliniensis. Taken together, our results demonstrate that the biological features and regulatory mechanisms of white-gray-opaque tristable transitions are largely conserved in the 2 pathogenic Candida species.

Candida parapsilosis is an emerging opportunistic pathogen, second in frequency only to C. albicans and commonly associated with both mucosal and systemic infections. Adhesion to biotic surfaces is a key step for the development of mycoses. The C. parapsilosis genome encodes 5 predicted agglutinin-like sequence proteins and their precise role in the adhesion process still remains to be elucidated. In this study, we focused on the putative adhesin Cpar2_404800, in view of its high homology to the most important adhesion molecule in C. albicans. Two independent lineages of C. parapsilosis CPAR2_404800 heterozygous and null mutants were obtained by site-specific deletion. CPAR2_404800 mutants did not differ from wild-type strain in terms of in vitro growth or in their ability to undergo morphogenesis. However, when compared for adhesion to a biotic surface, CPAR2_404800 null mutants exhibited a marked reduction in their adhesion to buccal epithelial cells (>60% reduction of adhesion index). Reintroduction of one copy of CPAR2_404800 gene in the null background restored wild type phenotype. A murine model of urinary tract infection was used to elucidate the in vivo contribution of CPAR2_404800. A 0.5 and 1 log10 reduction in colony forming unit numbers (per gram) was observed respectively in bladder and kidneys obtained from mice infected with null mutant compared to wild-type infected ones. Taken together, these findings provide the first evidence for a direct role of CPAR2_404800 in C. parapsilosis adhesion to host surfaces and demonstrate its contribution to the pathogenesis of murine urinary candidiasis.

Background: To identify the microorganisms that can grow rapidly in total parenteral nutrition (TPN) solutions, we investigated the growth of the major causes of catheter-related blood stream infection (Staphylococcus aureus, Serratia marcescens, Bacillus cereus, and Candida albicans) in TPN solutions containing lipid. Methods: The pH value of a TPN solution containing lipid (pH 6.0, containing 20 ppm of NaHSO3) was adjusted by the addition of HCl to 5.7, 5.4, or 4.9. The pH value of another TPN solution (pH5.5, containing 400 ppm of NaHSO3) was adjusted by the addition of NaOH to 5.9, 6.3, or 6.8. A specific number of each microorganism was added to 10 mL of each test solution and incubated at room temperature. The number of microorganisms was counted as colony forming units at 0, 24, and 48 hrs later. Results: C albicans increased similarly at any pH values in the TPN solution. The bacterial species also increased rapidly at pH6.0 in the solution containing 20 ppm of NaHSO3, but growth was suppressed as the pH value was reduced, with growth halted at pH4.9. However, these bacterial species did not increase, even at pH5.9, in the other solution containing 400 ppm of NaHSO3. Conclusions: These results suggest that Candida species can grow rapidly in almost all TPN solutions regardless of the acidity, lipid, and NaHSO3; also, some bacterial species may grow in TPN solutions containing lipid unless the pH value is 5.0 or less. Therefore, each TPN solution should be examined whether or not the bacterial species can proliferate.

Made available in DSpace on 2015-08-26T19:19:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-01. Added 1 bitstream(s) on 2015-08-31T13:04:47Z : No. of bitstreams: 1 S1517-83822014000400030.pdf: 624666 bytes, checksum: f2e052bac7537ce2986bb27ce433b6f4 (MD5) Made available in DSpace on 2015-08-26T19:19:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-01. Added 1 bitstream(s) on 2015-08-31T13:04:47Z : No. of bitstreams: 1 S1517-83822014000400030.pdf: 624666 bytes, checksum: f2e052bac7537ce2986bb27ce433b6f4 (MD5) Made available in DSpace on 2015-08-26T19:19:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-01. Added 1 bitstream(s) on 2015-08-31T13:04:47Z : No. of bitstreams: 1 S1517-83822014000400030.pdf: 624666 bytes, checksum: f2e052bac7537ce2986bb27ce433b6f4 (MD5) Made available in DSpace on 2015-08-26T19:19:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-01. Added 1 bitstream(s) on 2015-08-31T13:04:47Z : No. of bitstreams: 1 S1517-83822014000400030.pdf: 624666 bytes, checksum: f2e052bac7537ce2986bb27ce433b6f4 (MD5) Made available in DSpace on 2015-08-26T19:19:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-01. Added 1 bitstream(s) on 2015-08-31T13:04:47Z : No. of bitstreams: 1 S1517-83822014000400030.pdf: 624666 bytes, checksum: f2e052bac7537ce2986bb27ce433b6f4 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilm production of different species among the different sources of isolation: blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilm production was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilm production by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilm production was more frequent among bloodstream isolates than other clinical sources,in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilm production. The numbers of tandem-repeat copies per allele were not associated with biofilm production, suggesting the evolvement of other genetic determinants. Universidade Estadual Paulista Júlio de Mesquita Filho Instituto de Biociências de Botucatu Universidade Estadual Paulista Júlio de Mesquita Filho Faculdade de Medicina de Botucatu Universidade Estadual Paulista Júlio de Mesquita Filho Instituto de Biociências de Botucatu Universidade Estadual Paulista Júlio de Mesquita Filho Faculdade de Medicina de Botucatu FAPESP: 2007/01946-4 FAPESP: 2008/10835-4 FAPESP: 2008/09231-7

The incidence of fungal infections due to C. parapsilosis and closely related cryptic species (-psilosis complex) has increased in the last few years, but differences in virulence among these species have not been widely studied. Fifteen clinical isolates of C. parapsilosis, C. orthopsilosis, and C. metapsilosis, including the type strains, were used to evaluate their virulence in Galleria mellonella larvae. Fluctuations in the hemocyte density and in the phagocytic activity were also tested. Differences in the median survival for these species were demonstrated at 37 °C (2.6 ± 1.02, 2.3 ± 0.92, and 4.53 ± 1.65 d for C. parapsilosis, C. orthopsilosis, and C. metapsilosis, respectively). Galleria mellonella hemocytes phagocytosed C. metapsilosis strains more effectively than did for C. orthopsilosis and C. parapsilosis (P

Twenty samples of herbal soaps were evaluated for their antimicrobial activity against bacteria and yeast of significance in skin infections with the aim to provide some justification for the continued use of the soaps in the management of superficial skin infections. All the soaps were found to possess antimicrobial activity in a concentration and organism dependent manner. The soaps were more active against the gram positive organismsthan the Gram negative organisms while none of the soaps had activity against the tested yeasts. Only 35% of the soaps were appropriately packaged with adequate directions for use and storage. The study showed that the tested soaps possessed antimicrobial properties and they can contribute to the treatment and management of skin infections caused by bacteria if well prepared with the appropriate plant materials to target specific causative organisms and packaged with appropriate directions for use and storage.Keywords: Antimicrobial activity; Skin infections; Herbal soaps; Medicinal plants.

Ethanolic crude extracts prepared from the arils and seeds, pericarp, peels and from the whole fruit of Punica granatum, known as pomegranate, had their antifungal activity tested against Candida spp. The ethanolic crude extracts were analyzed by Mass Spectrometry and yielded many compounds such as punicalagin and galladydilacton. The extracts from the pericarp and peel showed activity against Candida spp., with MICs of 125 µg/mL. The effect of pericarp and peel extracts upon the morphological and structure of C. albicans and C. krusei were examined by scanning and transmission electron microscopy, with the visualization of an irregular membrane and hyphae, formation of vacuoles and thickening of the cell wall. The data obtained revealed potential antimicrobial activity against yeasts cells of the Candida genus, and the bioactive compounds could be responsible for changes in cell morphology and structure. The data obtained open new perspectives for future research in continuation to this study, where information such as determination of the site of action of the compounds could contribute to an alternative therapy against these organisms.

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi:10.1007/s00449-011-0545-8, 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268–1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate. Electronic supplementary material The online version of this article (doi:10.1007/s00449-011-0546-7) contains supplementary material, which is available to authorized users.

The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgarTM Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans.

Ching-Hsuan Lin works in the field of Candida biology. In this mSphere of Influence article, he reflects on how the papers “Use of ichip for high-throughput in situ cultivation of uncultivable microbial species” by D., Ching-Hsuan Lin works in the field of Candida biology. In this mSphere of Influence article, he reflects on how the papers “Use of ichip for high-throughput in situ cultivation of uncultivable microbial species” by D. Nichols, N. Cahoon, E. M. Trakhtenberg, L. Pham, et al. (Appl Environ Microbiol 76:2445–2450, 2010, https://doi.org/10.1128/AEM.01754-09) and “A new antibiotic kills pathogens without detectable resistance” by L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al. (Nature 517:455–459, 2015, https://doi.org/10.1038/nature14098) made an impact on him by inspiring him to explore new bioactive antimicrobial compounds with his collaborators.

Triglycerides, steryl esters, resin acids, free fatty acids and sterols are lipophilic extractives of wood (commonly referred to as pitch or wood resin) and have a negative impact on paper machine runnability and quality of paper. Thus, enzymes capable of modifying these compounds would be potential tools for reducing pitch problems during paper manufacture. In this work, 19 commercial lipase preparations were tested for their ability to degrade steryl esters, which may play a significant role in the formation and stabilisation of pitch particles. Six lipase preparations were shown to be able to degrade steryl esters. Lipase preparations of Pseudomonas sp., Chromobacterium viscosum and Candida rugosa were shown to have the highest steryl esterase activities. The enzymes were able to hydrolyse steryl esters totally in the presence of a surfactant (Thesit). Up to 80% of the steryl esters were degraded in aqueous dispersion. Preliminary characterisation of the enzymatic activities revealed that the lipase preparation of Pseudomonas sp. could be the most potential enzyme in industrial applications. The steryl esterase activity of this preparation was stable over a broad pH range and the enzyme was able to act efficiently at pH 6–10 and at temperatures up to 70 °C.

Background Candida africana is distributed worldwide and colonized in human genitalia and cause mainly vulvovaginal candidiasis (VVC). We report the multilocus sequence typing (MLST) analysis of C. africana from VVC. Methods MLST analysis of 43 strains of C. africana, which were isolated from vaginal specimens of patients with VVC, was performed. The enzymatic activity of phospholipase, esterase and haemolysis enzyme production was evaluated.The level of virulent genes and resistant genes mRNA expression was determined by using real-time PCR. Antifungal susceptibilities of the isolates were assayed by using the broth microdilution method. The statistical of the results was determined by the T test and Pearson chi-squared test. Results The MLST analysis revealed a substantial degree of genetic homogeneity. The DST782 and DST182 were the main MLST genotypes in C. africana. All the patients were symptomatic and with a high mycological cure rate when treated with commonly used antifungal agents.There were statistically significant differences in biofilm formation and phospholipase activity between C. africana and C.albicans. The level of virulent genes and resistant genes mRNA expression was higher in fluconazole-resistant strains. All C. africana isolates were susceptible to fluconazole, itraconazole, voriconazole, caspofungin, and micafungin. These isolates also exhibited low MICs to amphotericin B, flucytosine, and posaconazole. Conclusions Candida africana appear to be with a low level of sequence variation in MLST loci. Candida africana, a lower virulence candida, is susceptible to commonly used antifungal agents. This paper was presented at the conference of 8th Trend in Medical Mycology (6–9 October 2017, Belgrade, Serbia) and was published on conference abstract. Electronic supplementary material The online version of this article (10.1186/s12879-019-4071-7) contains supplementary material, which is available to authorized users.

C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host’s immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host’s environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.

In this paper we analyze an experiment for the use of low-cost gas sensors intended to detect bacteria in wounds using a non-intrusive technique. Seven different genera/species of microbes tend to be present in most wound infections. Detection of these bacteria usually requires sample and laboratory testing which is costly, inconvenient and time-consuming. The validation processes for these sensors with nineteen types of microbes (1 Candida, 2 Enterococcus, 6 Staphylococcus, 1 Aeromonas, 1 Micrococcus, 2 E. coli and 6 Pseudomonas) are presented here, in which four sensors were evaluated: TGS-826 used for ammonia and amines, MQ-3 used for alcohol detection, MQ-135 for CO2 and MQ-138 for acetone detection. Validation was undertaken by studying the behavior of the sensors at different distances and gas concentrations. Preliminary results with liquid cultures of 108 CFU/mL and solid cultures of 108 CFU/cm2 of the 6 Pseudomonas aeruginosa strains revealed that the four gas sensors showed a response at a height of 5 mm. The ammonia detection response of the TGS-826 to Pseudomonas showed the highest responses for the experimental samples over the background signals, with a difference between the values of up to 60 units in the solid samples and the most consistent and constant values. This could suggest that this sensor is a good detector of Pseudomonas aeruginosa, and the recording made of its values could be indicative of the detection of this species. All the species revealed similar CO2 emission and a high response rate with acetone for Micrococcus, Aeromonas and Staphylococcus.

Cultures are negative in ∼50% of invasive candidiasis. Data are emerging for the performance of nonculture tests such as mannan/antimannan, Candida albicans germ tube antibody, 1,3-β- d -glucan, PCR, and the T2Candida panel in diagnosing both candidemia and deep-seated candidiasis. In most settings, positive predictive values of nonculture test are low, and negative predictive values are high. For tests to be useful, clinicians must understand the pretest likelihood of invasive candidiasis and test performance for the most common disease manifestation in a given patient. This paper reviews nonculture Candida diagnostics and discusses how they might be used effectively in patient care.

Background and aims. Bacterial vaginosis (BV) is one the more frequently identified genital syndrome among childbearing aged women. The basic condition that generates this condition is a modification in the vaginal microbiota. The aim of this paper is to briefly review the current status of the art of BV and to report the results of a pilot study performed with an innovative PCR based technique. Materials and Methods. 36 samples of vaginal fluid routinely submitted for the diagnosis of BV to the Unit of Microbiology – GRHL were comparatively evaluated by standard techniques and with the HP-Vaginiti e Vaginosi NLM kit that simultaneously detects in a quantitative way specific DNA from Candida (albicans, glabrata; krusei, tropicalis), Gardnerella vaginalis, Lactobacillus spp. and Atopobium vaginae. Results and conclusions. Candida spp. has been identified in 8 samples with culture and in 15 with the molecular test. 29 G. vaginalis were found by PCR whereas only in 7 samples a specific prescription for this microbe was present (of which 4 positive). A. vaginae has been identified in 20 samples by the molecular approach and Lactobacillus spp. was identified in 19 samples (by culture) and in 32 by PCR. The overall diagnosis of BV was made in 9 patients by standard techniques and in 7 by applying the molecular approach. (Cohen’s kappa test: 0,84). The findings of this study clearly demonstrate that the joint use of the routine culture- based techniques with the multiplex PCR methods amplifies by far the sensitivity of the overall diagnostic workflow of BV.

This paper examines the process of selenium bioaccumulation and selenium metabolism in yeast cells. Yeast cells can bind elements in ionic from the environment and permanently integrate them into their cellular structure. Up to now, Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica yeasts have been used primarily in biotechnological studies to evaluate binding of minerals. Yeast cells are able to bind selenium in the form of both organic and inorganic compounds. The process of bioaccumulation of selenium by microorganisms occurs through two mechanisms: extracellular binding by ligands of membrane assembly and intracellular accumulation associated with the transport of ions across the cytoplasmic membrane into the cell interior. During intracellular metabolism of selenium, oxidation, reduction, methylation, and selenoprotein synthesis processes are involved, as exemplified by detoxification processes that allow yeasts to survive under culture conditions involving the elevated selenium concentrations which were observed. Selenium yeasts represent probably the best absorbed form of this element. In turn, in terms of wide application, the inclusion of yeast with accumulated selenium may aid in lessening selenium deficiency in a diet.

The microbial contamination of central air conditioning system is one of the important factors that affect the indoor air quality. Actual measurement and analysis were carried out on microbial contamination in central air conditioning system at a venue in Dalian, China. Illumina miseq method was used and three fungal samples of two units were analysed by high throughput sequencing. Results showed that the predominant fungus in air conditioning unit A and B were Candida spp. and Cladosporium spp., and two fungus were further used in the hygrothermal response experiment. Based on the data of Cladosporium in hygrothermal response experiment, this paper used the logistic equation and the Gompertz equation to fit the growth predictive model of Cladosporium genera in different temperature and relative humidity conditions, and the square root model was fitted based on the two environmental factors. In addition, the models were carried on the analysis to verify the accuracy and feasibility of the established model equation.

In the present paper, facile synthesis of Ag@ZnO core-shell nanocomposites is reported where zinc oxide is coated on biogenic silver nanoparticles synthesized using Andrographis paniculata and Aloe vera leaf extract. Structural features of as synthesized nanocomposites are characterized by UV-visible spectroscopy, XRD, and FTIR. Morphology of the above core-shell nanocomposites is investigated by electron microscopy. As synthesized nanocomposite material has shown antimicrobial activity against Candida krusei, which is an opportunistic pathogen known to cause candidemia. The possible mode of activity of the above material has been studied by in-vitro molecular techniques. Our investigations have shown that surface coating of biogenic silver nanoparticles by zinc oxide has increased its antimicrobial efficiency against Candida krusei, while decreasing its toxicity towards A431 human epidermoid carcinoma cell lines.

Candida spp. form a part of human and animal oral cavity flora. However Candida spp. is the main cause of dental related stomatitis in humans and stomatitis in dogs. Stomatitis treatment implies the use of azoles and polyenes to which yeasts build up resistance. The research is directed to the use of natural compounds such as essential oils. The aim of this paper is to define the antifungal activity of thyme oil on 15 clinical strains of Candida spp., isolated from humans and dogs and to determine if there is a difference in susceptibility between human and dog isolates. Sampling in patients with stomatitis was done by swabbing the denture or oral mucosa swab while sampling in dogs was done by swabbing the oral cavity mucosa after stomatitis has been diagnosed. In order to investigate the antifungal activity of thyme oil in vitro, microdilution method was used. Thyme oil expressed antifungal effects on all investigated strains. Also, our data show that the values of minimum fungicide concentration (MFC) and minimum inhibitory concentration (MIC) are lower in human strains. Explanation is that in most cases, stomatitis in humans is asymptomatic and thus not treated, so Candida strains have not developed resistance. On the other hand, stomatitis in dogs is followed by a marked clinical picture and treated is by antimicotics (mostly by azoles), therefore resistant Candida strains are more likely to occur., Candida spp. je sastavni deo mikrobioma usne duplje čoveka i psa. Međutim Candida spp. predstavlja glavni uzročnik proteznog stomatitisa kod čoveka i stomatitisa kod psa. Terapija stomatitisa podrazumeva korišćenje azola i poliena na koje poslednjih godina gljivice razvijaju otpornost. Istrživanja se usmeravaju ka primeni prirodnih preparata kao što su etarska ulja. Cilj rada je utvrdi antifungalnu aktivnost ulja timijana na kliničke izolate Candida spp. čoveka i psa, i utvrdi da li postoji razlika u osetljivosti sojeva Candide spp. izolovanih kod čoveka i psa. Uzorkovanje kod pacijenata sa stomatitisom je vršeno brisom proteze ili brisom sluzokože. Kod pasa je uzrokovanje vršeno brisom sluzokože usne duplje kada je dijagnostikovan stomatitis. Zasejani sojevi su in vitro tretirani korišćenjem mikrodilucione metode da bi se utvrdilo da li ulje timijana deluje na kliničke izolate Candida spp. čoveka i psa. Rezultati pokazuju da su vrednosti minimalne fungicidne koncentracije (MFC) i minimalne inhibitorne koncentracije (MIC) manje kod čoveka. Stomatitis je kod ljudi najčešće je asimptomatski, pacijenti se ne podvrgavaju lečenju, pa sojevi Candida spp. nisu razvili rezistenciju na antifungicidne preparate. Kod pasa stomatitis je praćen burnijom kliničkom slikom, leči se antimikoticima (uglavnom azolima) pa je verovatnoća pojave rezistentnih sojeva Candida spp. veća.

International audience; In this paper, we evaluate a rapid and safe pretreatment procedure using glass beads for MALDI-TOF yeast identification in a routine clinical laboratory avoiding the use of formic acid. We created a new yeast database library using 1186 yeasts, including 11 references strains. The database was tested using 2131 clinical isolates allowing accurate species-level identification in 98.9% (2107/2131) of cases with a score over 1.9 and in 99% (2123/2131) of the strains at the genus level. The new protocol is a rapid, reliable and safe procedure for the accurate identification of pathogenic Candida strains and requires minimal handling.

The design of new biocatalysts is a target that is receiving increasing attention. One of the most popular reactions in this regard is the Diels-Alder cycloaddition due to its applications in organic synthesis and the absence of efficient natural enzymes that catalyze it. In this paper, the possibilities of using the highly promiscuous Candida Antarctica lipase B (CALB) as a protein scaffold to re-design a Diels-Alderase has been explored by means of theoretical quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations. Free energy surfaces have been computed for two reactions in the wild-type and in several mutants with hybrid AM1/MM potentials with corrections at M06-2X/MM level. The study of the counterpart reactions in solution has allowed performing comparative analysis that render interesting conclusion. Since the dienophile anchors very well in the oxyanion hole of all tested protein variants, the slight electronic changes from reactant complex to the transition state suggest that mutations should be focused in favoring the formation of reactive conformations of reactant complex that, in turn, would reduce the energy barrier. This work was supported by the Spanish Ministerio de Economía y Competitividad (project CTQ2012-36253-C03), Universitat Jaume I (project P1•1B2014-26), Generalitat Valenciana (project PROMETEOII/2014/022), the Polish Ministry of Science and Higher Education ("Iuventus Plus" program project 0478/IP3/2015/73, 2015-2016) and the USA National Institute of Health (project NIH R01 GM065368). Authors acknowledge computational resources from the Servei d’Informàtica of Universitat Jaume I.

The use of biologic mesh, which is considered resistant to infection, has become common. It is preferred over synthetic mesh for use in contaminated fields. Fungal infection with infiltration of biologic mesh is rare and has not been reported. In this paper, we report a case of a patient who underwent multiple laparotomies and received multiple antibiotics and an azole antifungal. Biologic mesh was used, but it ultimately required removal because of chronic infection with Candida krusei. On biopsy, the yeast was found to have infiltrated the mesh.

The spectrum of Candida species associated with invasive fungal infections is evolving. New microbiology diagnostic tools, the increasing number of immunosuppressed patients with invasive devices and the use of prophylaxis with fluconazole could contribute to this phenomenon (1). In recent years, an increasing number of rare species with reduced susceptibility to antifungal molecules have been described, including C. ciferrii, C. inconspicua, C. guilliermondii, C. humicola, C. lambica, C. lipolytica, C. norvegensis, C. palmioleophila, C. rugosa, C. valida, C. fermentati, and C. lusitaniae (2)-(5). Data from the SENTRY Antimicrobial Surveillance Program-Fungal Objective (5) concerning bloodstream infections from 2008 and 2009 show that 4.5% of 348 episodes from 10 centers in Latin America were caused by species other than C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. In 1984, Lavarde et al. (6) reported the first clinical isolate of C. haemulonii from a blood culture. Since then, rare cases of human infections with C. haemulonii have been reported worldwide, including central venous catheter (CVC)-related bloodstream infections in patients from Argentina, Korea, and China (7)-(11); in preterm neonates receiving parenteral nutrition in Kuwait (12); and in a 37-year-old French patient with osteomyelitis of the left hallux (13). This pathogen has not been identified in previous reports of candidemia from Brazil (14)-(17). The present paper reports for the first time a case of fungemia caused by C. haemulonii in a tertiary hospital in the city of Sao Paulo. Clinical features and laboratory analyses, including phenotypic and molecular identification and antifungal susceptibility testing (AST), are described.

The incidence of Candida bloodstream infection has increased over the past years. In the Center-West region of Brazil, data on candidemia are scarce. This paper reports a retrospective analysis of 96 cases of Candida bloodstream infection at a Brazilian tertiary-care teaching hospital in the state of Mato Grosso do Sul, from January 1998 to December 2006. Demographic, clinical and laboratory data were collected from medical records and from the hospital's laboratory database. Patients' ages ranged from three days to 92 years, with 53 (55.2%) adults and 43 (44.8%) children. Of the latter, 25 (58.1%) were newborns. The risk conditions most often found were: long period of hospitalization, utilization of venous central catheter, and previous use of antibiotics. Fifty-eight (60.4%) patients died during the hospitalization period and eight (13.7%) of them died 30 days after the diagnosis of candidemia. Candida albicans (45.8%) was the most prevalent species, followed by C. parapsilosis (34.4%), C. tropicalis (14.6%) and C. glabrata (5.2%). This is the first report of Candida bloodstream infection in the state of Mato Grosso do Sul and it highlights the importance of considering the possibility of invasive Candida infection in patients exposed to risk factors, particularly among neonates and the elderly. RESUMO A incidência de infecções na corrente sangüínea causada por Candida spp. tem aumentado nos últimos anos. Na região Centro-Oeste do Brasil, os dados sobre candidemia são escassos. Realizamos uma análise retrospectiva de casos de infecção na corrente sangüínea por Candida em um hospital terciário de ensino de Mato Grosso do Sul. Noventa e seis episódios diagnosticados de janeiro de 1998 a dezembro de 2006 foram incluídos no estudo. Os dados demográficos e clínicos foram obtidos de prontuários; os dados laboratoriais provieram de registros do laboratório hospitalar. Dos pacientes, 43 (44,8%) eram crianças e 53 (55,2%) adultos, com idades variando de três dias a 92 anos. Das crianças, 25 (58,1%) eram recém-nascidas. As condições de risco mais encontradas foram: prolongado tempo de internação, uso de cateter venoso central e uso prévio de antibióticos. Cinqüenta e oito (60,4%) pacientes foram a óbito durante a hospitalização e oito (13,7%) deles foram a óbito 30 dias após o diagnóstico de candidemia. Candida albicans (45,8%) foi a espécie mais prevalente seguida por C. parapsilosis (34,4%), C. tropicalis (14,6%) e C. glabrata (5,2%). Esta é a primeira descrição de infecção na corrente sangüínea por espécies de Candida em Mato Grosso do Sul, confirmando a importância da suspeita clínica de infecções invasivas por tais microrganismos na evolução de pacientes expostos a fatores de risco, principalmente no caso de idosos e neonatos.

In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanol, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and 1-propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K(i)(I) were changed to "K(i)", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.

Using the (near) complete genome sequences of the yeastsCandida albicans, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, we address the evolution of a unique genetic code change, which involves decoding of the standard leucine-CTG codon as serine in Candida spp. By using two complementary comparative genomics approaches, we have been able to shed new light on both the origin of the novel Candida spp. Ser-tRNACAG, which has mediated CTG reassignment, and on the evolution of the CTG codon in the genomes of C. albicans,S. cerevisiae, and S. pombe. Sequence analyses of newly identified tRNAs from the C. albicans genome demonstrate that the Ser-tRNACAG is derived from a serine and not a leucine tRNA in the ancestor yeast species and that this codon reassignment occurred ∼170 million years ago, but the origin of the Ser-tRNACAG is more ancient, implying that the ancestral Leu-tRNA that decoded the CTG codon was lost after the appearance of the Ser-tRNACAG. Ambiguous CTG decoding by the Ser-tRNACAG combined with biased AT pressure forced the evolution of CTG into TTR codons and have been major forces driving evolution of the CTN codon family in C. albicans. Remarkably, most of the CTG codons present in extant C. albicans genes are encoded by serine and not leucine codons in homologous S. cerevisiae and S. pombe genes, indicating that a significant number of serine TCN and AGY codons evolved into CTG codons either directly by simultaneous double mutations or indirectly through an intermediary codon. In either case, CTG reassignment had a major impact on the evolution of the coding component of the Candidaspp. genome.[Supplemental material is available online at http://www.genome.organd at http://www.bio.ua.pt/genomica/Lab/Genomedata.html. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N. Federspiel.]

We have previously shown that cycloheximide resistance can be induced in a strain of Candida maltosa by modifying ribosomes (M. Takagi, S. Kawai, Y. Takata, N. Tanaka, M. Sunairi, M. Miyazaki, and K. Yano, J. Gen. Appl. Microbiol. 31:267-275, 1985). The present paper describes the cloning of the gene involved in this resistance (designated RIM-C for ribosome modification by cycloheximide) by using a host-vector system of Saccharomyces cerevisiae.