Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens

Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

In vitro evaluation of hydrolytic enzyme activity and biofilm formation of Candida parapsilosis species complex from a nosocomial environment

Abstract INTRODUCTION: Candida parapsilosis complex species, frequently found in hospital environments, have gained importance as etiological agents of candidemia. METHODS: Candida parapsilosis complex isolates from a nosocomial environment were identified and their hydrolitic enzyme activity and ability to form biofilm were characterized. RESULTS: Twenty-two C. parapsilosis sensu stricto isolates produced proteinase and three produced phospholipase. Most Candida metapsilosis isolates produced proteinase and one also produced phospholipase. All 29 isolates formed biofilms. CONCLUSIONS: The nosocomial environment may act as a reservoir for C. parapsilosis complex isolates with phenotypic features that could possibly lead to nosocomial infections and health complications in hospital patients.

Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics

Abstract Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs) and extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions. .

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida / Atividade diferencial in vitro de fosfolipases e proteinases ácidas de isolados clínicos de Candida

INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3 percent) of isolates tested were phospholipase positive and 16 (44.4 percent) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7 percent). Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001) and among the strains from different sites of origin (p=0.014). Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2 percent). Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901). CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

INTRODUÇÃO: Candida são leveduras comensais, porém, se o equilíbrio da flora normal for interrompido ou as defesas imunitárias estiverem comprometidas, espécies de Candida podem causar manifestações de doença. Vários atributos contribuem na virulência e patogenicidade de Candida, inclusive a produção de enzimas extracelulares hidrolíticas, especialmente fosfolipases e proteinases. O objetivo deste estudo foi verificar a atividade in vitro de fosfolipases e proteinases ácidas em isolados clínicos de Candida spp. MÉTODOS: Oitenta e dois isolados provenientes de pacientes hospitalizados coletados a partir de sítios de origem diversos foram analisados. A produção de fosfolipase foi verificada em meio egg yolk e a de proteinase em meio contendo soro albumina bovina. O estudo foi feito em triplicata. RESULTADOS: Cinquenta e seis (68,3 por cento) dos isolados testados apresentaram atividade de fosfolipase positiva e 16 (44,4 por cento) foram positivos para atividade de proteinase. C. tropicalis foi a espécie que apresentou o maior número de isolados positivos para fosfolipases (91,7 por cento). Diferenças estatisticamente significantes em relação à produção de fosfolipases entre as espécies e entre as cepas provenientes de diferentes sítios de origem foram detectadas. Quanto à produção de proteinases ácidas, os isolados de C. parapsilosis testados foram os maiores produtores (69,2 por cento). Entre as espécies analisadas, a porcentagem de produção de proteinase entre os isolados não diferiu estatisticamente (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901). CONCLUSÕES: A maioria dos isolados de C. não-albicans, assim como os de C. albicans, foram grandes produtores de enzimas hidrolíticas e, consequentemente, podem ser capazes de causar infecção em condições adequadas.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity / Diferenças na produção de exoenzimas e habilidade de aderência entre isolados de espécies de Candida provenientes do cateter, sangue e cavidade bucal

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1 percent and 55.9 percent of Candida albicans and 69.8 percent and 37.7 percent of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.

A produção de proteinase e fosfolipase e habilidade de adesão à célula epitelial bucal de 112 isolados de Candida originadas da cavidade bucal de pacientes infectados pelo HIV e de sangue e cateter de pacientes hospitalizados foram investigados. A produção de proteinase foi detectada por inoculação em ágar soro albumina bovina e a atividade de fosfolipase foi realizada usando emulsão de gema de ovo. A suspensão de levedura de cada isolado foi incubada com célula epitelial e o número de leveduras aderidas a célula epitelial foi contada. Uma porcentagem de 88,1 e 55,9 por cento de C. albicans e 69,8 e 37,7 por cento de isolados de Candida não albicans produziram proteinase e fosfolipase, respectivamente. Candida não albicans obtidas do cateter foram mais proteolíticos que isolados de Candida albicans (p < 0,001). Isolados do sangue foram mais proteolíticos do que isolados do cateter e cavidade bucal, enquanto isolados da cavidade bucal produziram mais fosfolipase do que aqueles isolados do sangue e cateter. C. albicans isoladas da cavidade bucal e do cateter foram mais aderentes à célula epitelial bucal do que isolados de Candida não albicans, mas não houve diferença na adesão entre os três locais analisados. Os resultados indicaram diferenças na produção de fosfolipase e proteinase e na habilidade de adesão à célula epitelial bucal entre os isolados de Candida das diferentes fontes. Este estudo sugere que a patogenicidade de Candida spp pode estar correlacionada ao local infectado.

Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology / Viability, taxonomic confirmation and enzymatic detection of Acremonium species preserved under mineral oil in the URM Culture Collection

Enzimas hidrolíticas secretadas por fungos têm um papel importante na patogenicidade das infecções. Objetivando avaliar a atividade enzimática foram testados 31 isolados de Acremonium mantidos na Coleção de Culturas University Recife Mycology. Fragmentos das culturas foram transferidos para caldo glicosado para reativação e posterior crescimento em meio ágar batata dextrose, para verificar viabilidade, pureza e confirmação taxonômica pela observação das características macroscópicas e microscópicas. Para detecção enzimática foram utilizados substratos de caseína do leite e gelatina para protease, amido para amilase e lecitina de soja para fosfolipase. Das 31 culturas, 26 (83,9 por cento) mantiveram-se viáveis e 24 (92,3 por cento) foram confirmadas taxonomicamente. Das 24 culturas, 12 (50 por cento) apresentaram atividade proteásica, duas (16,7 por cento) em caseína do leite, uma (8,3 por cento) em gelatina e nove (75 por cento) em ambos os substratos; 16 (66,7 por cento) degradaram amido. Nenhuma cultura apresentou atividade fosfolipásica. Conclui-se que espécies de Acremonium são capazes de produzir enzimas envolvidas na patogenicidade das infecções fúngicas.

Hydrolytic enzymes secreted by fungi play an important role in the pathogenesis of infection. With the aim of evaluating the enzymatic activity, 31 isolates of Acremonium stored in the University of Recife Mycology (URM) Culture Collection were tested. Culture fragments were transferred to glycoside broth for reactivation and further growth in potato dextrose agar medium in order to investigate viability and purity and to confirm the taxonomy through observing the macroscopic and microscopic characteristics. To detect enzymes, milk casein and gelatin were used as substrates for proteinase, starch for amylase and soy lecithin for phospholipase. Among the 31 cultures, 26 (83.9 percent) remained viable and 24 (92.3 percent) were confirmed taxonomically. Out of these 24 cultures, 12 (50 percent) presented proteinase activity, of which two (16.7 percent) were on milk casein, one (8.3 percent) on gelatin and nine (75 percent) on both substrates; 16 (66.7 percent) degraded starch. None of the cultures presented phospholipase activity. It was concluded that Acremonium species are able to produce enzymes that are involved in the pathogenicity of fungal infections.

Phenotypic aspects of oral strains of Candida albicans in children with down's syndrome / Aspectos fenotípicos de cepas de Candida albicans orais em crianças com síndrome de down

The aim of this article is to characterize the biological aspects of oral strains of C. albicans in children with Down's syndrome. These yeasts were analyzed as to their macromorphological and enzymatic aspects and were tested as to their in vitro susceptibility to antifungal drugs using broth microdilution to determine the minimum inhibitory concentration (MIC). The morphotyping revealed that all oral C. albicans isolates from children with Down's syndrome promoted the formation of fringes regardless of size, while the control group presented smaller fringes. All oral C. albicans strains produced proteinase, but those with phospholipolytic activity showed greater enzyme capacity in the test group. In vitro susceptibility showed that all oral C. albicans isolates were sensitive to the drugs used.

O objetivo deste artigo foi caracterizar os aspectos biológicos de cepas de C. albicans orais em crianças com síndrome de Down. Estas leveduras foram analisadas quanto aos seus aspectos macromorfológicos e enzimáticos e foram testadas quanto a sua suscetibilidade in vitro a drogas antifúngicas, usando a microdiluição em caldo para a determinação da concentração inibitória mínima (CIM). A morfotipagem revelou que todos os isolados de C. albicans orais de crianças com síndrome de Down induziram à formação de franjas independente do tamanho, enquanto o grupo controle teve franjas menores. Todas as cepas de C. albicans orais produziram proteinase, mas aquelas com atividade fosfolipidolítica mostraram maior capacidade enzimática no grupo teste. A suscetibilidade in vitro mostrou que todos os isolados de C. albicans orais foram sensíveis a drogas empregadas.

Freqüência e atividade enzimática de Candida albicans isoladas da mucosa bucal de crianças de uma creche da prefeitura de Fortaleza / Frequency and enzymatic activity of Candida albicans isolated from the buccal mucosa of children of a day-care center of the city hall of Fortaleza, Ceará, Brazil

As candidíases bucais (também chamadas sapinhos) que ocorrem em crianças são causadas por uma deficiência imunológica, bem como por outros fatores tais como má higiene bucal e esterilização inadequada dos utensílios utilizados pelas mesmas, que potencializam a ocorrência dessa infecção fúngica. Considerando esse fato, foram avaliadas a freqüência e a atividade enzimática de Candida sp. isoladas em crianças de uma creche pública (Aprisco) na cidade de Fortaleza, Ceará. Foram coletadas amostras da mucosa bucal de 364 alunos de 1 a 5 anos de idade. Elas foram semeadas em ágar Sabouraud dextrose com cloranfenicol, incubadas por 72 horas a 37ºC e identificadas por testes micológicos. Verificou-se que 67 (18 por cento) apresentaram leveduras do gênero Candida. A Candida albicans foi a mais freqüente, com 30 isolados (45 por cento), seguida pelas C. tropicalis (31 por cento), C. guilliermondii (17 por cento), C. glabrata (4,5 por cento) e C. stellatoidea (1,5 por cento). Com relação às atividades enzimáticas das cepas de Candida albicans, 20 por cento produziram a enzima proteinase e 33 por cento, a fosfolipase. As Candida albicans isoladas da mucosa bucal de crianças dessa creche da prefeitura apresentaram uma fraca atividade enzimática. Assim, conclui-se que essas cepas parecem ter uma baixa virulência.

Enzimotipagem de espécies do gênero Candida isoladas da cavidade bucal / Enzymotiping of species of the genus Candida isolated from the oral cavity

Foram avaliadas quanto a produçäo de exoenzimas fosfolipase e proteinase, 79 amostras de Candida isoladas da cavidade bucal de pacientes com lesöes bucais características de candidose e indivíduos com boca clinicamente normal, atendidos na Faculdade de Odontologia de Ribeiräo Preto USP. Dentre as cepas de C. albicans isoladas de lesöes bucais, a fosfolipase e proteinase foram detectadas em, respectivamente, 83,3 por cento e 66,7 por cento. C. tropicalis e C. parapsilosis produziram somente proteinase. Quanto às cepas isoladas de nichos sem lesäo, do total de 32 C. albicans, 71,9 por cento apresentaram fosfolipase e 68,7 por cento proteinase. C. tropicalis apresentou apenas a enzima proteinase, C. glabrata, C. krusei, C. guilliermondii e Candida spp, näo apresentaram nenhuma das exoenzimas. Entre as amostras de C. albicans de ambos os grupos, o enzimotipo 22 (fosfolipase positiva e proteinase fracamente positiva) foi prevalente. Enzimotipos diferentes foram detectados em amostras da mesma espécie provenientes de mesmo paciente

Toxinas killer e produção de enzimas por Candida albicans isoladas da mucosa bucal de pacientes com câncer / Killer toxin and enzyme production by Candida albicans isolated from buccal mucosa in patients with cancer

Opportunistic infections of the oral cavity are primarily caused by Candida and frequently occur in patients with cancer who are undergoing chemotherapy and antibiotic treatment. Of the specimens received from the oral mucosa of 44 patients with cancer, 25 (56.8%) yielded Candida on culture in Sabouraud agar. Twenty four of these isolates were identified as C. albicans (96%) and 1 as C. krusei (4%). The phenotypic characteristics of these isolates showed that all of them were strongly proteolytic, had a high ability to produce phospholipase, and presented the byotypes characterized as 811 (95.8%) and 511 (4.2%) in terms of susceptibility to killer toxins.

Infecções oportunistas da cavidade bucal são primariamente causadas por fungos do gênero Candida e freqüentemente ocorrem em pacientes com câncer que estão sobtratamento quimioterápico e antibacteriano. De 44 amostras coletadas da mucosa oral de pacientes com câncer, observou-se o isolamento de 25 leveduras do gênero Candida em cultivo realizado em ágar Sabouraud-dextrose. Foram identificados Candida albicans em 24 (96%) isolados e C. krusei em 1 (4%). As características fenotípicas das amostras de Candida albicans mostraram que todos os isolados foram fortemente proteolíticos, capazes de produzir fosfolipases e possuíam os biotipos caracterizados como 811(95,8%) e 511 (4,2%) em relação a susceptibilidade às toxinas killer.

Serotyping, proteinase and phospholipase production associated with the pathogenicity of Candida albicans strains isolated from frog meat

Quinze amostras de Candida albicans isoladas de coxas de räs (Rana catesbeiana, Shaw, 1802) comercializadas no Brasil, foram estudadas quanto ao sorotipo e fatores de virulência "in vitro" e "in vivo". A sorotipagem revelou predominância do sorotipo B. Todas as amostras estudadas foram produtoras de fosfolipases e 66,6 por cento produziram proteinases. No estudo da patogenicidade foram observados dois tipos de alteraçöes renais: uma do tipo agudo com abscessos corticais e outra do tipo crônico com pielonefrite e envolvimento unilateral

Phospholipase A production by isolates of Salmonella species.

The incidence of intra- and extra-cellular phospholipase-A production by Salmonella isolates from human, poultry and environmental material was investigated. Nearly 17 per cent (15/90) of the strains tested produced phospholipase A. Phospholipase production was encountered in S. typhi, S. paratyphi A, S. typhimurium, S. seftenberg, S. bareilly, S. weltevredeen, S. newport, S. adelaide, S. alachua and S. gallinarum. Maximum (10/15) phospholipase producers were isolated from the human material. Phospholipase positive human isolates exhibited a high incidence of simultaneous multiple drug resistance, enterotoxigenicity and cell surface hydrophobicity.