Development of a bis-pyrene phospholipid probe for fluorometric detection of phospholipase A2 inhibition.

In this work, we report the design, synthesis, and application of a bis-pyrene phospholipid probe for detection of phospholipase A2 action through changes in pyrene monomer and excimer fluorescence intensities. Continuous fluorometric assays enabled detection of the activities of multiple PLA2 enzymes as well as the decrease in catalysis by PLA2 from honey bee venom caused by the inhibitor p-bromo phenacylbromide. Thin-layer chromatography and mass spectrometry analysis were also used to validate probe hydrolysis by PLA2. Mass spectrometry data also supported cleavage of the probe by phospholipase C and D enzymes, although changes in fluorescence were not observed in these cases. Nevertheless, the bis-pyrene phospholipid probe developed in this work is effective for detection of PLA2 enzyme activity through an assay that enables screening for inhibitor development.

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains.

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.

Comparative analysis of proteinase, phospholipase, hydrophobicity and biofilm forming ability in Candida species isolated from clinical specimens.

Candida species are the commensal organisms of human and animal mucosa that cause a wide range of debilitating diseases in immunocompromised patients and other susceptible individuals. The present study aimed to investigate the ability of clinical isolates of various Candida species to produce proteinase and phospholipase, hydrophobicity and biofilm forming ability that assumed to have a vital role in Candida pathogenicity. Eighty-four Candida strains belonged to Candida albicans (44.1%), C. glabrata (5.9%), C. guilliermondii (5.9%), C. krusei (10.8%), C. parapsilosis (26.2%), and C. tropicalis (7.1%) were examined for proteinase and phospholipase production, cell surface hydrophobicity and biofilm forming ability. The production of proteinase and phospholipase was detected in 81 (96.4%) and 79 (94.1%) of the strains, respectively. C. albicans showed the highest proteinase and phospholipase activity (mean Pz values of 0.42±0.25 and 0.72±0.28) and biofilm formation ability (0.66±0.22). C. parapsilosis had the highest hydrophobicity (42.97±16.1), which showed a good correlation with biofilm formation ability. A considerable percentage of non-albicans Candida strains produced significant amounts of proteinase and phospholipase with a good ability of biofilm formation in vitro. Taken together, our results further substantiated that enzymatic activity, hydrophobicity and the ability for biofilm formation are important virulence factors which may be account for pathogenicity of various Candida species distributed in albicans and non-albicans groups.

Activities of Endogenous Lipase and Lipolysis Oxidation of Low-Salt Lactic Acid-Fermented Fish (Decapterus maruadsi).

There is increasing demand for low-salt meat products that retain traditional flavors. In this study, dry-salted fish (Decapterus maruadsi) were processed by 2 methods to obtain traditional salted fish (HS) and low-salt lactic acid-fermented fish (LAF). The relationship between lipolysis and lipid oxidation was investigated by evaluating changes in endogenous lipase (lipolytic enzymes; lipoxygenase, LOX), free fatty acid composition, thiobarbituric acid reactive substances (TBARS), and peroxide value (POV) during processing. Lipolytic enzyme activity showed a decreasing trend, in general. LOX activity initially increased and eventually decreased. Phospholipase, acid lipase, and neutral lipase activity was 0.33, 0.17, 0.57 (in HS) and 0.39, 0.25, 0.67 (in LAF) times in the final product than the activity levels observed in fresh fish. A principal component analysis indicated that phospholipase and neutral lipase play major roles in promoting lipid hydrolysis (in HS and LAF), the correlation between lipolytic activity and lipid oxidation in HS is greater than the correlation in LAF, and the contribution of LOX to lipid oxidation was minor in salted fish.

Phenotypical characterization and molecular identification of clinical isolates of Candida tropicalis / Caracterización fenotípica e identificación molecular de aislamientos clínicos de Candida tropicalis

Background. Candida tropicalis is an increasingly important human pathogen which usually affects neutropenic oncology patients with common hematogenous seeding to peripheral organs and high mortality rates. Candida pathogenicity is facilitated by several virulence attributes, including secretion of hydrolytic enzymes; however, little is known regarding the C. tropicalis ability to secrete them and their role in the disease. Aims. To confirm by molecular means the identification of 187 clinical isolates (127 from blood, 52 from urine, and 8 from diverse clinical origins) phenotypically identified as C. tropicalis, and to investigate their in vitro aspartyl proteinase, phospholipase, esterase, hemolysin, DNase and coagulase activities. Methods. The molecular confirmation was performed by ITS sequencing, and the enzymatic determinations were conducted using plate assays with specific substrates, with the exception of coagulase, which was determined by the classical tube test. Results. The majority of the strains exhibited a very strong or strong activity of aspartyl proteinase, phospholipase and esterase. A 4.7% of the bloodstream isolates were hemolysin producers, and all were negative for the coagulase and DNase assays. Conclusions. Very strong activities of aspartyl proteinase, phospholipase and esterase profiles were detected, and a statistical association between phospholipase production and blood and urine isolates was found (AU)

Antecedentes. Candida tropicalis es un patógeno del ser humano cada vez más importante que afecta especialmente a pacientes oncológicos neutropénicos, en los cuales es frecuente la diseminación hematógena del microorganismo a órganos periféricos, lo que conlleva elevadas tasas de mortalidad. La patogenicidad de Candida es facilitada por diversos factores de virulencia, incluyendo la secreción de enzimas hidrolíticas; sin embargo, poco se sabe respecto a la habilidad de C. tropicalis para su secreción, así como el papel que desempeña en la enfermedad. Objetivos. Confirmar por un método molecular la identidad de 187 aislamientos clínicos (127 de sangre, 52 de orina y 8 de orígenes diversos) fenotípicamente identificados como C. tropicalis y estudiar la actividad in vitro de las enzimas proteinasa aspártica, fosfolipasa, esterasa, hemolisina, DNasa y coagulasa. Métodos. La confirmación molecular se llevó a cabo mediante secuenciación del ITS y las determinaciones enzimáticas se llevaron a cabo mediante ensayos en placa con sustratos específicos, a excepción de la coagulasa, que se determinó mediante la clásica prueba en tubo. Resultados. La mayoría de los aislamientos analizados mostraron un perfil de actividad muy fuerte o fuerte de proteinasa aspártica, fosfolipasa y esterasa. El 4,7% de las cepas sanguíneas fue productora de hemolisinas y todas fueron negativas para coagulasa y DNasa. Conclusiones. Se detectaron perfiles con una actividad proteinasa aspártica, fosfolipasa y esterasa muy fuerte entre los aislamientos clínicos analizados, así como también se encontró asociación estadística entre la producción de fosfolipasa y aquellos aislamientos obtenidos de sangre y orina (AU)

The frequency, antifungal susceptibility and enzymatic profiles of Candida species in cases of onychomycosis infection.

Although the frequency of candidal onychomycosis is increasing daily, there is little information in literature about the epidemiology, pathogenesis, and antifungal susceptibility of this dermatological disease. This study aimed to provide information about the epidemiology, pathogenesis, and azole susceptibility of Candida species isolated from patients living in a region with continental climate. After identification of the isolated strains using conventional methods, proteinase and phospholipase activities were determined by a plate method and biofilm-forming ability was determined using the microplate method. Susceptibility of the same species to fluconazole (FLU), voriconazole (VRC), miconazole (MNZ), itraconazole (ITZ), and ketoconazole (KTZ) were determined by microdilution method. The 50 Candida isolates included 23 C. parapsilosis (46%), 13 C. albicans (26%), 4 C. guilliermondii(8%), 4 C.tropicalis (8%), 2 C.krusei(2%), 1 C.lusitaniae (2%), 1 C. sake (2%), and 1 C. kefyr (2%) isolates. The geometric mean (GM) of the minimum inhibitory concentration (MIC) for FLU, KTZ, VRC, MNZ, and ITZ was 0.4 µg/mL, 0.08 µg/mL, 0.08 µg/mL, 0.2 µg/mL, and 0.6 µg/mL, respectively. Proteinase, phospholipase, and biofilm-forming ability were detected in 18%(9/50), 20%(10/50), and 6%(3/50) of the Candida isolates, respectively. We found that the most frequently isolated species is C.parapsilosis. On the basis of the GM values, the most effective azoles are ketoconazole and voriconazole. The isolated Candida species exhibited low phospholipase, proteinase, and biofilm formation activities.

Investigations of the prevalence and virulence of Candida albicans in periodontal and endodontic lesions in diabetic and normoglycemic patients.

OBJECTIVE: The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. MATERIAL AND METHODS: A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. RESULTS: C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). CONCLUSIONS: Candida albicans was isolated more frequently and had higher virulence in diabetic patients.

Investigations of the prevalence and virulence of Candida albicans in periodontal and endodontic lesions in diabetic and normoglycemic patients

Abstract Pulpal and periodontal tissues have similar microbiota that allows cross-contamination between the pulp and periodontal tissues. Objective The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. Material and Methods A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. Results C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). Conclusions Candida albicans was isolated more frequently and had higher virulence in diabetic patients.

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy / Caracterización enzimática de aislamientos clínicos y ambientales de Cryptococcus neoformans de Italia

Background. Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. Aims. In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. Methods. Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. Results. Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. Conclusions. The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too (AU)

Antecedentes. Cryptococcus neoformans es una levadura encapsulada que produce infecciones oportunistas. Los factores de virulencia involucrados en la patogénesis de la criptococosis incluyen la existencia y el tamaño de la cápsula polisacarídica, la producción de melanina por medio de la enzima fenoloxidasa, el crecimiento a 37°C y la secreción de ciertas enzimas como proteinasa, fosfolipasa y ureasa. Existen otras enzimas que son secretadas por C. neoformans, pero su papel en la virulencia de este hongo aún no es conocido. Objetivos. Se investigó la producción de fosfolipasa tanto en aislamientos de C. neoformans obtenidos de pacientes como de aislamientos recuperados de deposiciones de aves, y se comparó el grado de producción con el de la síntesis de la enzima fenoloxidasa. Además, distingue las cepas mediante la definición de la actividad de 19 enzimas extracelulares diferentes. Métodos. Se estudiaron 205 aislamientos clínicos de C. neoformans y 32 ambientales. La producción de fenoloxidasa se determinó por el crecimiento de colonias de color marrón en medio de Staib. Para determinar la actividad fosfolipasa extracelular se utilizó el método semicuantitativo en placa con yema de huevo. Con el método comercial API ZYM se determinó la producción de otras 19 enzimas extracelulares. Resultados. El análisis estadístico de los resultados mostró una producción de fosfolipasa significativamente mayor entre los aislamientos clínicos en comparación con los ambientales. No se encontraron diferencias significativas entre ambos grupos en la producción de fenoloxidasa. En lo referente a las 19 enzimas extracelulares valoradas mediante el sistema API ZYM, la fosfatasa ácida mostró la mayor actividad en ambos grupos. Respecto a la enzima α-glucosidasa, nuevamente los aislamientos clínicos presentaron una actividad significativamente mayor. Todos los aislamientos de ambos grupos presentaron actividad leucina-arilamidasa, si bien los aislamientos clínicos procesaron mayor cantidad de sustrato de manera significativa. Conclusiones. La mayor producción de enzima fosfolipasa entre los aislamientos clínicos evidencia que esta enzima puede estar implicada en la patogénesis de la criptococosis. También es interesante la actividad extracelular de las enzimas fosfatasa ácida, α-glucosidasa y leucina-arilamidasa, valorada por medio del sistema comercial API ZYM. Diversos estudios apuntan a que estas enzimas están implicadas en la virulencia de bacterias y parásitos; nuestros resultados muestran también su posible implicación como factores de virulencia en las infecciones por Cryptococcus (AU)

Usefulness of the Non-conventional Caenorhabditis elegans Model to Assess Candida Virulence.

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.

Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

INTRODUCTION: Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. AIMS: The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. PATIENTS AND METHODS: The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-ß, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. RESULTS: Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-ß=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and ß among all studied isolates were almost similar. CONCLUSION: In the present study, the prevalence of VVC among investigated women was higher than the previous report from Khuzestan but C. albicans has yet remained the predominant agent of VVC in this area. Given to the EAI, the virulence of C. albicans in VVC can be mediated by phospholipase, esterase and proteinases.

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy.

BACKGROUND: Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. AIMS: In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. METHODS: Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. RESULTS: Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. CONCLUSIONS: The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.

ß-Endorphin enhances the phospholipase activity of the dandruff causing fungi Malassezia globosa and Malassezia restricta.

ß-Endorphin is known to stimulate phospholipase production by Malassezia pachydermatis during canine dermatoses. The role of ß-endorphin in Malassezia infection in humans is not well studied. The present study compares the influence of ß-endorphin on Malassezia globosa and Malassezia restricta isolated from patients with seborrhoeic dermatitis/dandruff (SD/D) and healthy controls. Malassezia isolates (five each of the two species from patients and healthy controls) were grown on modified Dixon's agar with or without 100 nmol/L ß-endorphin. Phospholipase activity was quantified based on its ability to hydrolyze L-α-phosphatidylcholine dimyristoyl (phospholipid substrate). Free fatty acid was measured by a colorimetry method. In isolates from patients, the phospholipase activity significantly increased after exposure to ß-endorphin (M. globosa, P = .04; M. restricta, P = .001), which did not occur in isolates from healthy controls. Moreover, after ß-endorphin exposure the patient isolates had significantly higher (P = .0004) phospholipase activity compared to the healthy control isolates. The results suggest that isolates of M. globosa and M. restricta from patients may differ from those of healthy humans.

Virulence Attributes and Antifungal Susceptibility Profile of Opportunistic Fungi Isolated from Ophthalmic Infections.

Investigations of both virulence factors and antifungal susceptibility profiles are crucial for understanding the pathogenesis and prognosis of ophthalmic mycoses. In this study, we investigated the in vitro antifungal susceptibility of amphotericin B (AMB), voriconazole (VRC), and natamycin (NAT) against a set of 50 fungal isolates obtained from patients with ocular mycoses using the Clinical and Laboratory Standards Institute broth microdilution method. In addition, putative virulence factor, such as secretory phospholipases and proteinases, and biofilm formation activity were analyzed. The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of the antifungals across all isolates were the following (in increasing order): VRC (0.70 µg/mL), AMB (0.81 µg/mL), and NAT (1.05 µg/mL). The highest activity against 14 Aspergillus strains was exhibited by VRC (GM MIC: 0.10 µg/mL), followed by AMB and NAT (GM MICs: 0.21 and 0.27 µg/mL), respectively. However, for 12 Fusarium spp., the GM MIC of VRC (2.66) was higher than those of NAT and AMB (GM MICs 1.3 and 0.8 µg/mL, respectively). Proteinase and phospholipase activity were observed in 30 % and 42 % of the isolates, respectively, whereas only 8 % of the isolates were able to produce biofilms. Phospholipase activity was observed in all Fusarium isolates, but not in any of the Aspergillus isolates. In contrast, biofilm-forming capability was detected in 25 % of the Fusarium isolates, but none of the Aspergillus isolates. The differences in the MICs of AMB, VRC, and NAT, biofilm-forming ability and proteinase and phospholipase activities among the isolates were not significant (p > 0.05). Overall, our study suggests no significant correlation between the antifungal susceptibility profiles and virulence attributes of ocular fungal isolates.

Reliability of the agar based method to assess the production of degradative enzymes in clinical isolates of Candida albicans.

The aim of this study was to establish a reproducible protocol using the methodology of hyaline zones around the colonies on specific agar plates for phospholipase and proteinase production. This was an in vitro double-blind experiment, in which the dependent variables were the enzymatic activity measurements (Pz) for the production of phospholipase (Pz-ph) and the production of secreted aspartyl proteinases (Pz-sap). Three independent variables give rise to different measurement protocols. All measurements were carried out at two different moments by four examiners (E1, E2, E3, and E4). The minimum sample size was 30 Candida albicans clinical isolates. Specific agar plates for phospholipase and SAPs production were prepared according the literature. The intra-and inter-examiner reproducibility for each protocol was estimated using the Intraclass Correlation Coefficient (ICC) and its confidence interval (95% CI). Based on the results obtained for both phospholipase and SAPs, there appears to be no consensus on the protocol chosen for each particular examiner. Measuring the colonies in triplicate may be the main factor associated with the increase in measurement accuracy and should therefore take precedence over measuring only one colony. When only one examiner is responsible for taking measurements, a standard protocol should be put in place and the statistical calibration of this researcher should be done prior to data collection. However, if two or more researchers are involved in the assessment of agar plates, our results suggest that the protocols using software to undertake plate reading is preferred.

Trends in antifungal susceptibility and virulence of Candida spp. from the nasolacrimal duct of horses.

This was a cross-sectional study to investigate the antifungal susceptibility and production of virulence factors in strains of Candida isolated from the outlet and the lumen of the nasolacrimal duct of horses in the state of Ceará, Brazil. The samples were obtained from 103 horses. Sterile cotton swabs were used to collect the material from the outlet of the nasolacrimal duct and urethral probes, for the instillation of 2 ml of saline solution, were used to collect samples from the lumen of the nasolacrimal duct. A total of 77 Candida isolates were obtained, with C. famata, C. tropicalis, C. guilliermondii, and C. parapsilosis sensu lato as the most prevalent species. One isolate (C. glabrata) was resistant to caspofungin. One isolate was resistant only to fluconazole (C. parapsilosis sensu lato), 11 were resistant only to itraconazole (7 C. tropicalis, 2 C. guilliermondii, 1 C. famata, 1 C. parapsilosis sensu lato), while eight C. tropicalis showed resistance to both azoles. Overall, 28 isolates produced phospholipases and 12 produced proteases. These results highlight the importance of investigating the antifungal susceptibility and virulence trends of Candida spp. from the microbiota of the nasolacrimal duct of horses.

Histone deacetylases inhibitors effects on Cryptococcus neoformans major virulence phenotypes.

Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.

Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

Enzymatic and hemolytic activity in different Candida species / La actividad enzimática y hemolítica en diferentes especies de Candida

Background: Candida species, in conditions of microbiota imbalance or decreased immune defenses, may be one of the main human fungal pathogens. Virulence factors constitute the mechanisms used by the fungus to avoid host defenses. Aims: This study aimed to investigate the in vitro production of virulence factors, such as hemolytic activity, and deoxyribonuclease (DNase), proteinase, and phospholipase activities in Candida spp. Methods: Fifty clinical isolates were analyzed for virulence factors: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), and Candida krusei (5). Hemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture media containing, respectively, agar-base DNA, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. Results: Forty-eight (96%) of 50 isolates showed hemolytic activity, with 10 (20%) positive for DNase, 19 (38%) for proteinase, and 16 (32%) for phospholipase. Statistically significant differences were observed between species for phospholipase (p < 0.0001) and proteinase (p < 0.05) production. Conclusions: It is concluded that all species had hemolytic activity. DNase activity was detected in all species except in C. glabrata; proteinase activity was detected in C. albicans, C. tropicalis, and C. parapsilosis; and phospholipase activity was observed in C. albicans and C. tropicalis (AU)

Antecedentes: Las levaduras del género Candida, en condiciones de desequilibrio de la microbiota o de disminución de las defensas inmunológicas, pueden ser uno de los principales patógenos fúngicos del hombre. Los factores de virulencia constituyen los mecanismos utilizados por el hongo para evadir las defensas del huésped. Objetivos: Este estudio tiene como objetivo investigar la producción in vitro de algunos factores de virulencia, como la actividad hemolítica, y las actividades desoxirribonucleasa (DNasa), proteinasa y fosfolipasa en Candidaspp. Métodos: Se analizaron 50 aislamientos clínicos: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), y Candida krusei (5). La actividad hemolítica fue determinada en placas de agar glucosado de Sabouraud, con glucosa al 3% y un 7% de hematíes de oveja. Los medios de cultivo de agar-ADN, yema de huevo y albúmina bovina fueron utilizados para determinar las actividades DNasa, fosfolipasa y proteinasa, respectivamente. Resultados: De los 50 aislamientos, 48 (96%) presentaron actividad hemolítica, 10 (20%) fueron positivos para DNasa, 19 (38%) para proteinasa y 16 (32%) para fosfolipasa. Se observaron diferencias estadísticamente significativas entre las especies para las actividades fosfolipasa (p < 0,0001) y proteasa (p < 0,05). Conclusiones: Se concluye que todas las especies estudiadas poseen actividad hemolítica. La actividad DNasa fue detectada en todas las especies, excepto en Candida glabrata; la actividad proteinasa fue detectada en C. albicans, C. tropicalis y C. parapsilosis, y la actividad fosfolipasa se observó en C. albicans y C. tropicalis (AU)

Protease and phospholipase activities of Candida spp. isolated from cutaneous candidiasis / Actividades proteasa y fosfolipasa en aislamientos de Candida procedentes de candidiasis cutánea

Background: cases of superficial and invasive mycoses caused by emerging species of Candida have been increasingly reported over the last thirty years. The production of hydrolytic enzymes plays a central role in the fungal infective process. In Candida infections the secretion of both proteases and phospholipases are well-known virulence attributes. Aims: to determine the protease and phospholipase production from 58 human clinical isolates of Candida obtained from individuals with cutaneous candidiasis seen in the Human and Veterinary Diagnostic Mycology Sector from Universidade Federal Fluminense (UFF), Brazil, from November 2008 to August 2009. Methods: fungal identification was performed using biochemical tests. Proteolytic activity was detected on agar plates containing bovine serum albumin, and phospholipase production was determined on egg-yolk plates. Results: the Candida species isolated were Candida parapsilosis (27.59%), Candida famata (18.96%), Candida albicans (15.52%), Candida haemulonii (12.06%), Candida ciferri (8.62%), Candida guilliermondii (6.90%), Candida tropicalis (5.17%) and Candida lipolytica (5.17%). All isolates of C. albicans produced both protease and phospholipase. As regards the isolates of non-C. albicans Candida species, 53.06% and 4.08% were able to produce protease and phospholipase, respectively. For example, the majority of isolates of C. parapsilosis (15/16) produced protease, while 40% of C. ciferri isolates (2/5) were phospholipase producers. This study shows, for the first time, that C. ciferri and C. haemulonii strains were able to produce protease. Conclusions: collectively, our results showed that different species of Candida isolated from cutaneous lesions were able to produce proteases and/or phospholipases, which are multifunctional molecules directly involved in the infectious process of these fungi

Antecedentes: Los casos de micosis superficiales e invasoras relacionados con las especies emergentes de Candida se han reportado progresivamente durante las últimas tres décadas. La producción de enzimas hidrolíticas juega un papel central en varios contextos de la patogenicidad fúngica. Con respecto a la infección por Candida, la secreción de proteasas y fosfolipasas son atributos de virulencia bien conocidos. Objetivos: Determinar y comparar la producción de proteasa y fosfolipasa de 58 aislamientos clínicos humanos de diferentes especies de Candida obtenidos de pacientes con candidiasis cutánea, atendidos en el Sector de Diagnóstico Micológico Humano y Veterinario de la Universidad Federal Fluminense (UFF), durante el período de noviembre de 2008 a agosto de 2009. Métodos: La identificación de las especies de Candida se realizó mediante pruebas bioquímicas, la actividad proteolítica se detectó en placas de agar que contenían albúmina de suero bovino y la actividad fosfolipasa se determinó utilizando el método de la placa de yema de huevo semi-cuantitativa. Resultados: Las especies aisladas fueron Candida parapsilosis (27,59%), Candida famata (18,96%), Candida albicans (15.52%), Candida haemulonii(12,06%), Candida ciferri(8,62%), Candida guilliermondii(6,90%), Candida tropicalis (5,17%) y Candida lipolytica (5,17%). Todos los aislamientos de C. albicans produjeron tanto proteasa como fosfolipasa. El 53,06% de los aislamientos de Candida no-C. albicans fueron capaces de producir proteasa y el 4,08% fosfolipasa. La mayoría de los aislamientos de C. parapsilosis (15/16) produjo proteasa, mientras que el 40% de los aislamientos de C. ciferri (2/5) fueron productores de fosfolipasa. Se describe por primera vez en la literatura científica la producción de proteasas por cepas de C. haemulonii y C. ciferri. Conclusiones: Nuestros resultados muestran el potencial que tienen los aislamientos de Candida provenientes de lesiones cutáneas para producir proteasas y fosfolipasas (AU)