UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS CÂMPUS DE BOTUCATU SUSCEPTIBILIDADE ANTIFÚNGICA, PRODUÇÃO DE BIOFILME E CARACTERIZAÇÃO DO GENE ALS3 EM ISOLADOS DE Candida albicans E NÃO-albicans DO HOSPITAL DAS CLÍNICAS, UNESP, BOTUCATU ARIANE CRISTINA MENDES DE OLIVEIRA BRUDER NASCIMENTO BOTUCATU - SP 2009 �������� �� �� �������������������������� ������������� ��� ��� ��� ���������� ������� ���� ����� �� ��� ���� �� ��� !������ ������"����� ��� �#� ��� ��� �"��� #��� ���$ ������ � ��� UNIVERSIDADE ESTADUAL PAULISTA INSTITUTO DE BIOCIÊNCIAS CÂMPUS DE BOTUCATU SUSCEPTIBILIDADE ANTIFÚNGICA, PRODUÇÃO DE BIOFILME E CARACTERIZAÇÃO DO GENE ALS3 EM ISOLADOS DE Candida albicans E NÃO-albicans DO HOSPITAL DAS CLÍNICAS, UNESP, BOTUCATU ARIANE CRISTINA MENDES DE OLIVEIRA BRUDER NASCIMENTO ORIENTADOR: PROF. Dr. EDUARDO BAGAGLI BOTUCATU - SP 2009 �������� �� � ���������� ��� ���������� ��� ������������� ��� ��� ��� ���������� ������� ���� ����� �� ��� ���� �� ��� !������������"���������#������� �"��� #��� ���$ ������ � ��� FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉCNICA DE AQUISIÇÃO E TRATAMENTO DA INFORMAÇÃO DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CAMPUS DE BOTUCATU – UNESP BIBLIOTECÁRIA RESPONSÁVEL: Selma Maria de Jesus Bruder-Nascimento, Ariane Cristina Mendes de Oliveira. Susceptibilidade antifúngica, produção de biofilme e caracterização do gene ALS3 em isolados de Candida albicans e não-albicans do Hospital das Clínicas, UNESP, Botucatu / Ariane Cristina Mendes de Oliveira Bruder Nascimento – Botucatu : [s.n.], 2009. Dissertação (mestrado) – Universidade Estadual Paulista, Instituto de Biociências, Botucatu, 2009. Orientador: Eduardo Bagagli Assunto CAPES: 40300005 1. Candida albicans 2. Infecção hospitalar 3. Biofilme (Microbiologia) CDD 616.969 Palavras-chave: ALS3; Candida albicans; Candida parapsilosis; Biofilme; Susceptibilidade antifúngica � �� �������� � �� ��������� ���� �������� ������ ����������������� ������������ � �� � �� ��������� � �� � ������ ����� ��� ������������������� (Francis Thompson, 1859-1906) � �� ���� ���� ���� ���� ���� �������� ���������� ���������� ���������� ������ ���� ���� ���� ���� ���� ���� ���� ���� È com muito amor e gratidão que dedico este estudo à Ana Luiza Panhoza, pelo apoio, amor e dedicação inigualáveis. !���� �������� � ���� �� �� ������ �������"� ���"� ���� ��#$� (O Pequeno Príncipe - Antoine de Saint-Exupéry)���� � �� ���� �% �����&�'�()��)*�����)�% �����&�'�()��)*�����)�% �����&�'�()��)*�����)�% �����&�'�()��)*�����)���� ���� Agradeço especialmente... ...ao Prof. Dr. Eduardo Bagagli, por aceitar a orientação deste estudo e conduzir seu desenvolvimento, com muita sabedoria e paciência; ...a meu marido, Thiago Bruder Nascimento, pelo companheirismo, amor e paciência a mim oferecidos durante a elaboração deste trabalho, por todos os momentos de alegria e pelas palavras de conforto e apoio; ...à Terue Sadatsune, por sua força, conhecimento e disposição, diante das minhas limitações.� � ���� �% �����&�'�()�% �����&�'�()�% �����&�'�()�% �����&�'�()���� Foram muitos, os que me ajudaram a concluir este trabalho. Meus sinceros agradecimentos... ...à Ana Luiza Panhoza, pelas oportunidades, pois, sem sua ajuda, nada teria sido possível; ...a meu pai, Luís Antônio de Oliveira (in memorian), e meu avô, Orlando Luis de Oliveira (in memorian), que, mesmo de outro plano, iluminam meu caminho; ...à minha mãe, Maria Mendes, e minha avó Ercília Oliveira, pela educação que me foi oferecida; ...às minhas irmãs, Aruana Passarelli e Ariah Oliveira, e minha sobrinha, Ana Luíza Passarelli, pelas quais supero todas as dificuldades na tentativa de passar a elas bons exemplos a serem seguidos. ...aos meus tios, Paulo e Ana Luiza Panhoza, Fabio e Mara Carmello, José Luiz e Alessandra Oliveira, Cláudia Oliveira e Marcello Pazzetto, pelos olhares sempre atentos, pelos conselhos sempre sensatos e também pela confiança, amizade e exemplo; ...aos meus primos, Lucas e Luiza Oliveira, Bianca e Victória Carmello, pela confiança, amizade e pelos momentos de alegria; ...a Thiago Antunes do Nascimento, por me ensinar que o amor e a paciência são ferramentas essenciais para a educação e formação, mas principalmente pela sua doçura e pureza. ...aos meus cunhados, Bruno Passarelli e Thatiane Bruder, pelo apoio; ...a meu padrastro, Antônio Carlos de Souza, pelo apoio; ...aos amigos, Carlos Camargo, Sandra Bosco, Virginia Richini, Raquel Cordeiro e Severino Assis Marcoris, pelo interesse e pela colaboração � ���� indispensável, disponibilizando informações técnicas essenciais ao bom andamento deste trabalho. ...às amigas, Raquel Pires de Campos, Sandra Olbrich, Keila Zamboni e Natália Godinho, pelo carinho, pelo apoio profissional e emocional. ...à amiga Talita Pimentel e família, amigos que me auxiliaram muito e que me lembrarei com carinho pela vida toda; ...aos professores Maria Fátima Sugizaki, Augusto Cezar Montelli e Vera Rall, pelas valiosas sugestões; ...a Sílvio e Emília Nascimento, Cemiro e Milva Bruder, Rachel Nascimento, Sílvia e Ivaldo dos Santos, pelo apoio e amizade; ...aos docentes e funcionários do departamento de Microbiologia e Imunologia, IBB-UNESP que participaram deste trabalho; ...aos funcionários do Laboratório de Microbiologia do Hospital das Clínicas da Faculdade de Medicina de Botucatu, pela disposição. ...enfim, a todos que diretamente ou indiretamente me ajudaram neste trabalho. � �� Suporte financeiro processo n º�2007/01946-4 � � � � � � � � � � � � � !(� �� �� ���+� � �� ��������,��###$� (O Pequeno Príncipe - Antoine de Saint-Exupéry) � �� SUMÁRIO Página Resumo ..................................................................................................... 01 Introdução ................................................................................................ 05 Justificativa e Objetivos ....................................................................... 11 Referências bibliográficas .................................................................... 12 Capítulo 1: Species distribution and susceptibility profile of Candida species in a Brazilian Public Tertiary Hospital ………………………… 19 Referências bibliográficas .................................................................... 26 Tabelas ………………………………………………………………. 32 Capítulo 2: Biofilm production and ALS3 central domain polymorphism in Candida species from different clinical sources …………………...... 34 Referências bibliográficas .................................................................... 43 Tabelas e Figuras ……………………………………………………. 46 Considerações Finais e Conclusões ……………………………………. 53 � �� RESUMO Leveduras oportunistas do gênero Candida são capazes de disseminar-se em hospedeiros susceptíveis, num processo crescente nos últimos anos. Um fator complicador destes quadros ocorre quando estas leveduras são capazes de produzir biofilme, principalmente quando associadas a cateteres ou outros dispositivos médicos, elevando o poder de penetração e invasão em órgãos do hospedeiro. Por também conferir maior resistência às drogas antifúngicas do que as células dispersas, o biofilme fúngico tornou-se um dos maiores problemas no combate a estas infecções. A base genética da produção de biofimes nestas leveduras é complexa, porém já foi determinado o envolvimento de genes da família ALS, codificadores de glicoproteínas de adesão. Dentre os oito genes desta família (ALS1 ao ALS7 e ALS9), destaca-se o papel de ALS3. O gene ALS3, assim como todos os outros genes da família, apresenta uma estrutura composta por 3 domínios. O domínio 5’, região bem conservada; um domínio central que apresenta motifs de 108pb repetidos em tandem, com variações de tamanho entre os genes da mesma família e entre o mesmo gene em diferentes espécies, em uma mesma espécie e até mesmo entre alelos de uma mesma cepa, e o domínio 3, menos conservado que o domínio 5’, que pode apresentar variações de tamanho e de algumas seqüências de aminoácidos. Tendo em vista a crescente incidência de infecções por esse microrganismo em todo o mundo, o presente estudo objetivou investigar a freqüência das diferentes espécies de Candida em nossa região e caracterizá-las quanto à susceptibilidade a drogas antifúngicas e produção de biofilme, e possível correlação da produção de biofilme com polimorfismos de tamanho do gene ALS3. Os resultados obtidos confirmam a crescente incidência de espécies não-albicans, principalmente isoladas de infecções invasivas como cultura de sangue e liquido peritoneal, onde C. parapsilosis foi a espécie mais freqüente isolada. Em relação à produção de biofilme, também os isolados de infecções invasivas apresentaram maior positividade para a produção de biofilme que os de secreção vaginal e urina. Quanto às espécies, amostras de Candida não-albicans também apresentaram maior positividade para a produção de biofilme que as de C. albicans. Reações da polimerase em cadeia foram realizadas para o gene ALS3, polimorfismos de tamanho foram detectados, mas a correlação do padrão de polimorfismos com a produção, in-vitro, de biofilme não foi significativa. O presente estudo também apresenta dados de susceptibilidade a quatro drogas antifúngicas, fluconazol, cetoconazol, itraconazol e anfotericina B, onde todas apresentaram excelente atividade sobre as cepas testadas e resistência foi detectada em poucos isolados. Os dados obtidos no presente trabalho podem refletir a situação das infecções por Candida spp. em nossa região, bem como orientar no tratamento e prevenção de infecções. � �� ABSTRACT Opportunistic yeasts of the genus Candida are able to disseminate into the bloodstream in susceptible hosts, in an increasing course in the recent years. A complicating factor is when these yeasts are capable of producing biofilms, especially associated with catheters or other medical devices. Biofilm also confers greater resistance to antifungal drugs than dispersed cells, so the fungal biofilm has become one of the greatest problems in combating these infections. The genetic basis of the biofim production by yeasts is complex, but it has been know the involvement of ALS gene family, encoders of adhesion glycoproteins. Among the eight genes of this family (ALS1 to ALS7 and ALS9), the ALS3 are considered the most important. The ALS3 gene, such as the others members of the family, have three general domains: the 5’domain, conserved, with approximately 1300-pb; followed by a central domain consisting entirely of tandem-repeats of a 108-pb sequence, that are somewhat variable; and the 3’ domain, which is least conserved in length and sequence. Considering the increase incidence of these infections worldwide, the aims of this study were identify the frequency of Candida species in our region, to characterize the profile of antifungal susceptibility; to quantify the biofilm production and to correlate this production with the ALS3 gene length polymorphism. Our data confirm the increase incidence of non-albicans species, mainly when obtained from invasive infections, such as blood and peritoneal fluid, in which C. parapsilosis was the most frequent isolated species. The same was also observed to biofilm production, in which isolates obtained from invasive infections (blood and peritoneal fluid) are more biofilm producers than that obtained from vaginal secretion and urine. Among the different species, isolates of non-albicans also are more biofilm producers than C. albicans. Polimerase chain reactions are used to evaluate the ALS3 gene length polymorphism. The number of tandem- repeats copies varied from seven to fourteen in the C. albicans isolates, and presented no correlation with biofilm production. The study also presents data of susceptibility tests against fluconazole, itraconazole, ketoconazole and amphotericin B. All these drugs showed a greatest activity, and resistance was observed in a few numbers of isolates. These data appear to reflect the real situation of Candida infection in the Brazilian public tertiary hospital, and might serve as guide for better treatment and prevention strategies. � �� INTRODUÇÃO Fungos patogênicos, especialmente espécies de Candida, têm emergido como importantes agentes de infecções oportunistas, principalmente, em indivíduos com a imunidade comprometida, incluindo aidéticos, pacientes com câncer submetidos à quimioterapia, transplantados em terapia imunossupressora e pacientes com diabetes avançada (RICHARDSON, 2005; APERIS et al., 2006). Acredita-se que a maioria dos casos de candidemias seja adquirida por via endógena, pela translocação do patógeno através do trato digestivo, local de rica colonização por Candida spp. (COLE, HALAWA, ANAISSIE, 1996; NUCCI, ANAISSIE, 2001). No entanto, estas infecções também podem ser adquiridas por via exógena, através do contato das mãos de profissionais da saúde com pacientes portadores de cateter, implante de próteses contaminadas, bem como pela administração parenteral de soluções contaminadas (PFALLER, 1995; WENZEL, 1995, TROFA, GÁCSER, NOSANCHUCK, 2008). Acredita-se que a maior parte das candidemias é sempre precedida pelo evento colonização pela mesma espécie de levedura, o que é considerado um importante fator de risco para o desenvolvimento destas infecções (COLE, HALAWA, ANAISSIE, 1996; NUCCI , ANAISSIE, 2001). Os principais fatores que predispõem os pacientes a infecções disseminadas incluem a colonização do trato gastrointestinal por espécies de Candida, resultante do uso prolongado de agentes antibacterianos de amplo espectro, ruptura da mucosa gastrointestinal por agentes citotóxicos, e neutropenia. Entretanto, o cateter venoso central, aparenta ser o fator de risco mais comum para o desenvolvimento de candidemia em pacientes não-neutropênicos ou que não apresentam imunodeficiência (REX, 1996). A partir da década de 80, a incidência de candidemias aumentou substancialmente em hospitais terciários de todo o mundo. (WISPLINGHOFF et al., 2004), e atualmente essas infecções têm emergido como os maiores responsáveis pela morbidade e mortalidade nos pacientes imunodeprimidos (PFALLER et al., 2000, 2008). No Brasil, Colombo et al. (2007) conduziram um estudo epidemiológico reunindo dados sobre infecções de corrente sanguínea documentados em quatro hospitais da cidade de São Paulo. Um total de 7038 episódios de bacteremias e fungemias ocorridos no período de um ano foi avaliado, Candida spp. responderam por 4,3% do total das infecções de corrente sanguínea. Freqüência semelhante foi também detectada no Hospital das Clínicas da Faculdade de Medicina de Botucatu (HC/FMB), que de um total de 6417 episódios e amostras de culturas positivas avaliadas, no período de janeiro de 1991 a dezembro de 1994, Candida spp. foram isoladas em 222 (3,5%) culturas, as � � quais foram oriundas principalmente das unidades de pediatria e berçário (SUGIZAKI et al., 1998). Ruiz et al. (2005) relataram que, nesse mesmo hospital, as espécies mais freqüentes foram C. albicans (38,7%) e C. parapsilosis (30,7%). Embora a C. albicans seja a principal espécie isolada de pacientes com fungemia (PFALLER et al., 1998a,b,c; SANDVEN et al., 1998; KRCMÉRY JR V, KOVACICOVÁ G, 2000), têm aumentado os relatos de infecções causadas por espécies não-albicans (LACAZ et al., 2002; COLOMBO et al., 2007). Em 1963, eram conhecidas apenas cinco espécies de Candida causadoras de doenças humanas, C. albicans, C. parapsilosis, C. tropicalis, C. stellatoidea e C. guilliermondii. Atualmente são conhecidas cerca de 20 espécies de Candida implicadas em micoses superficiais ou invasivas em humanos (DIGNANNI, SOLOMKIN, ANAISSIE, 2003). As principais espécies de interesse clínico são: C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, C. guilliermondii e C. lusitaniae. Entretanto, número progressivo de casos relacionados a espécies emergentes de Candida tem sido descrito, envolvendo isolamentos de C. dubliniensis, C. kefyr, C. rugosa, C. famata, C. utilis, C. lipolytica, C. norvegensis, C. inconspicua entre outras (COLEMAN et al., 1998). A freqüência de espécies de Candida não-albicans é depende da população de pacientes estudada, da terapêutica utilizada, do uso de antibióticos ou outras medidas adotadas (PFALLER, 1995; ABI-SAID et al., 1997; NUCCI, COLOMBO, 2007; PFALLER et al., 2008). Paralelamente ao aumento das infecções causadas por leveduras do gênero Candida, especialmente a nível hospitalar, tem sido observado o aparecimento de resistência aos antimicóticos, assim como a seleção de espécies não-albicans. Há alguns anos, considerava-se que os fungos eram regularmente sensíveis aos antimicóticos, apenas algumas espécies Candida podiam adquirir resistência à 5-fluorcitosina (DIASSIO, BENETT, MYERS , 1978), observando-se mais tarde o surgimento de resistência de C. albicans em pacientes com candidíase granulomatosa crônica sob tratamento prolongado com cetoconazol (HORSBURGH, KIRKPATRICK, 1983). Até esse momento, o problema não parecia ter maior repercussão, mas uma década mais tarde a situação mudaria drasticamente ao observar-se um aumento na freqüência de candidemias devido não apenas à C. albicans resistentes, mas também para espécies diferentes da C. albicans, geralmente menos suscetíveis aos antifúngicos. Entre estas últimas, destacavam a C. krusei que é intrinsicamente resistente ao fluconazol, e C. glabrata, cuja suscetibilidade aos azóis é muito variável (NGUYEN et al., 1996; PFALLER et al., 2000; REX, RINALDI, PFALLER, 1995). Porém, mais notórias foram as falhas de tratamento observadas a partir de 1985 em pacientes com AIDS e mucosite � � candidiásica sob tratamento com fluconazol, que desenvolveram candidíase orofaringeana crônica refratária à terapia (REVANKAR et al., 1998; NG, DENNING, 1993; SANGEORZAN et al., 1994). Diferentemente dos países da América do Norte, onde a emergência de espécies não- albicans parece estar associada à pressão seletiva do uso do fluconazol, no Brasil as espécies não-albicans mais prevalentes são sensíveis a esta droga (REX et al., 2001). Na ultima década, é crescente o numero de trabalhos documentando alterações na susceptibilidade das leveduras do gênero Candida às drogas antifúngicas. Embora muitos sejam os trabalhos relatando que a maioria dos isolados apresenta-se sensível ao fluconazol, já foi observado o desenvolvimento de resistência em pacientes previamente expostos aos azóis, seja por uso profilático ou indiscriminado da droga em determinadas populações (NOLTE et al., 1997; SAFDAR et al., 2001; KERSUN et al., 2008). Muitos programas de vigilância vêm documentando dados da distribuição das espécies e perfis de susceptibilidade às drogas. Consideráveis variações vêm sendo demonstradas entre diferentes hospitais ou diferentes países a respeito da incidência das espécies como etiologia de infecções e perfis de susceptibilidade dos microrganismos isolados. Em países desenvolvidos, existem acordos entre as instituições para unir dados epidemiológicos a fim de confirmar a magnitude das infecções, principalmente de corrente sanguínea, por Candida spp., bem como os perfis de susceptibilidade a drogas, gerando um importante banco de dados sobre as tendências das infecções e características mais freqüentes de cada espécie, o que não acontece na América Latina, onde esses estudos geralmente são limitados a uma única instituição (PFALLER et al., 1998a,b,c; RANGEL-FRAUSTRO et al., 1999; PFALLER et al., 2008). Essa situação é menos agravante em nosso país, onde já foi conduzido um grande estudo sobre candidemias, envolvendo 11 instituições, o qual apresentou consideráveis taxas de morbidade e mortalidade, embora a presença de amostras resistentes aos antifúngicos tenha sido rara (COLOMBO et al., 2006). O National Committe for Clinical Laboratory Standards (NCCLS) dos Estados Unidos, denominado, a partir de 2005, Clinical and Laboratory Standards Institute (CLSI) publicou um método de referência para testes de susceptibilidade antifúngica em leveduras, o M27-A2. Trata-se de um método quantitativo, que contem técnicas de diluição em meio líquido, para se determinar a concentração inibitória mínima (CIM), em leveduras, frente à anfotericina B, 5- fluorcitosina e derivados azólicos, incluindo cetoconazol, fluconazol, itraconazol, voriconazol, além de posaconazol e ravuconazol, estes últimos ainda não comercializados no Brasil (CLSI, Documento MA 27-A2). � �� Assim como acontece com bactérias, já foram relatados casos de infecção por fungos multi-resitentes, como por exemplo, algumas cepas de C. glabrata, que por sua vez ocorrem em 20-24% das candidemias nos EUA (RICHARDSON, 2005; PFALLER, DIECKEMA, MERZ, 2007). A multi-resistência a drogas é um sério fator complicador no tratamento de infecções fúngicas oportunistas que, frequentemente, ocorrem nos pacientes imunocomprometidos (THAKUR et al., 2008). Entre os atributos relacionados com o potencial patogênico da C. albicans, bem como de outras leveduras do mesmo gênero, está a incrível capacidade de adesão destes microrganismos (CALDERONE, BRAUN, 1991; RAMAGE et al., 2005; TROFA, GÁCSER, NOSANCHUCK, 2008), podendo levar à formação de biofilme. A capacidade de formação do biofilme pode ser considerada um potente fator de virulência, podendo estar presente em todas as espécies de Candida (REX, 1996). Como os biofilmes geralmente são mais resistentes aos mecanismos de defesa do hospedeiro e às drogas antimicrobianas do que as células dispersas, eles representam um fator predisponente de infecção para muitos pacientes (BAILLIE, DOUGLAS, 1998; DONLAN, 2001). A formação de biofilme em C. albicans vem sendo descrita como um processo gradual que se inicia com a aderência a um substrato, seja ao próprio tecido do hospedeiro ou ao dispositivo médico, resultando na formação de uma confluente camada basal de células que se dividem e produzem hifas, como projeções tubulares direcionadas para a região superior do biofilme (SOLL, 2008). Estas células durante o desenvolvimento do biofilme produzem uma matriz extracelular estável de substâncias poliméricas (DOUGLAS, 2003; CHANDRA et al., 2001; SOLL, 2008). O estágio final do desenvolvimento do biofilme é a maturação, quando ocorre menor crescimento das leveduras e elevado crescimento das hifas, nesta fase ocorre o envolvimento do biofilme pela matriz extracelular. A estimulação da produção da matriz extracelular durante o desenvolvimento do biofilme de C. albicans ainda é de causa desconhecida, o que se sabe, no entanto, é que a composição da matriz inclui: carboidratos, proteínas, fósforo, glicose e hexosaminas, mas a maior parte desse conteúdo ainda não foi identificada (BAILLIE, DOUGLAS, 2000; BLANKENSHIP, MITCHELL, 2006). Em resumo, os diferentes estágios que fazem parte do processo de desenvolvimento do biofilme incluem: estágio da aderência, célula-substrato e célula-célula; estágio de formação e desenvolvimento das hifas; e estágio de maturação, onde ocorre a produção da matriz extracelular que vai envolver e proteger as células do biofilme. Em estudo recente sobre quorum-sensing, Blankenship, Mitchell (2006) sugeriram um novo estágio no ciclo de vida do � �� biofilme de C. albicans, o estágio da dispersão, no qual células filhas se desenvolvem como células não aderentes, podendo ser facilmente liberadas do biofilme. Diversos estudos vêm confirmando que a produção de biofilme por espécies não- albicans é significativamente mais freqüente do que em C. albicans (SHIN et al., 2002; TUMBARELLO et al., 2007). Em C. parapsilosis, a produção de biofilme constitui-se em importante fator de virulência. C. parapsilosis é bem conhecida como causadora de fungemia e candidíase invasiva associada à hiperalimentação parenteral, dispositivos intravasculares e soluções oftálmicas contaminadas (PLOUFFE et al., 1977; SOLOMON et al., 1984; O’DAY, HEAD, ROBINSON, 1987; WEMMS et al., 1987; WEMMS, 1992). Vários fatores dão à C. parapsilosis uma vantagem seletiva, incluindo a capacidade de proliferar-se em altas concentrações de glicose e de aderência a materiais protéticos (CRITCHLEY, DOUGLAS, 1985; WEMMS et al., 1987). Pfaller, Messer, Hollis (1995), estudaram a produção de biofilme por amostras clínicas de C. parapsilosis crescidas em meio de cultura contendo glicose e verificaram que as isoladas de sangue e de cateter apresentaram maior produção de biofilme do que amostras de outros sítios anatômicos. A base molecular da formação e do desenvolvimento do biofilme destes fungos ainda não está completamente compreendida, porém, já está bem estabelecida que a interação da C. albicans com as células do hospedeiro ou superfícies inertes resulta em alterações na expressão de diferentes genes. Diferentes estudos têm descrito mudanças nos níveis de expressão gênica durante o desenvolvimento do biofilme (MARCHAIS et al., 2005, MURILLO et al., 2005). Estudos sobre a base genética da produção de biofilme têm se beneficiado significativamente com os avanços recentes observados na biologia molecular e genômica. Isto tem sido particularmente observado com a C. albicans, cujo genoma já foi totalmente seqüenciado (JONES et al., 2004; ODDS, BROWN, GOW, 2004; BRAUN et al.,, 2005; http://candida.bri.nrc.ca). C. albicans é uma levedura que se desenvolve na forma diplóide, com o material genético organizado em 8 cromossomos (1-7 e cromosssomo R), e o genoma haplóide é constituído de 14.851 kb (kilobases), contendo 6.419 ORFs (open reading frame, sequências de leituras, ou seja, genes codificantes) com mais de 100 codons de tamanho e 224 introns, (JONES et al., 2004; BRAUN et al., 2005; ODDS et al., 2007). Sistemas ou plataformas de microarray também já foram desenvolvidos para C. albicans, o que permite analisar a expressão gênica de todos os genes simultaneamente, nas diversas condições fisiológicas (CAO et al., 2005; http://genome.wustl.edu/ activity/ma/calbicans/). O envolvimento de alguns genes no processo de formação e desenvolvimento do biofilme parece já estar bem estabelecido (RAMAGE et al., 2005; NOBILE, MITCHELL � � 2006). Dentre os vários grupos de genes implicados neste fenótipo, constatou-se que os da família ALS (agglutinine like sequence) presentes em C. albicans e espécies relacionadas desempenham papel chave neste processo, por codificar proteínas com características de glicoproteínas de adesão à superfície da célula (HOYER, PAYNE, HECHT, 1998; HOYER, 1998). Já foi demonstrado que genes ALS estão com sua expressão aumentada durante a formação do biofilme (CHANDRA et al., 2001; GARCIA-SANCHEZ et al., 2004; GREEN et al., 2004; O’CONNOR et al., 2005). O gene ALS1 em C. albicans foi descrito pela primeira vez por Hoyer et al. (1995) e, desde então, pesquisadores vêm tentando entender sua relação com o restante da família ALS e explorando suas proteínas e funções. A família ALS presente em C. albicans inclui oito genes (ALS1-ALS7 e ALS9) que codificam muitas glicoproteínas de superfície (HOYER et al., 2008). Cada gene da família ALS apresenta uma estrutura similar composta por três domínios: um dominio 5’, na extremindade N, composto por 1299 a 1308pb, que apresenta 55-90% de similaridade entre os diferentes genes da família; um domínio central variável, organizado em tandem repeats, com motifs de 108pb que se repetem ao longo do domínio; e um domínio 3’, extremidade C, que é relativamente variável em tamanho e seqüência de nucleotídeos entre os genes da mesma família (HOYER, HECHT, 2001). Os genes da família ALS estão localizados em três cromossomos distintos: ALS1, ALS2, ALS4, ALS5 e ALS9 estão localizados no cromossomo 6, ALS6 e ALS7 estão localizados no cromossomo 3, e ALS3 no cromossomo R (HOYER et al., 1995; HOYER, PAYNE, HECHT, 1998; HOYER, HECHT 2001). O tamanho de um mesmo gene ALS freqüentemente varia dentro de uma mesma espécie e entre alelos de uma mesma cepa devido a diferenças no número de cópias dos motifs de 108pb organizados em tandem repeat, presentes no domínio central de cada gene (HOYER, HECHT, 2001). É comum, por exemplo, uma mesma cepa apresentar padrões diferentes (duas bandas) para o gene ALS1 devido à variabilidade do número de repetições dos motifs na região do domínio central em cada alelo (HOYER, 2001). Os genes ALS exibem diversos níveis de variabilidade, incluindo espécie-específica e alelo-específica, diferenças de tamanho para um mesmo gene, diferenças na regulação gênica espécie-específica, ausência de um gene ALS particular em certos isolados, e regiões codificadoras adicionais em outros (HOYER, HECHT, 2001). Estudos moleculares sobre a expressão de genes de ALS demonstraram que os mesmos são regulados e expressos diferencialmente em função de processos fisiológicos celulares, tais como o estágio de crescimento e morfologia da célula, ou seja, predominantemente leveduriforme ou na forma de hifas e pseudo-hifas (HOYER et al., 1995; HOYER et al., 1998; HOYER, PAYNE, � �� HECHT,1998). Constatou-se que destes genes, o ALS1, que codifica glicoproteínas de superfície celular, apresenta-se em alta expressão em células do biofilme de C. albicans (GARCÍA-SÁNCHEZ et al., 2004). O gene ALS3 também mostrou alta expressão, porém, aparentemente associado à produção de hifas de C. albicans (HOYER et al., 1998). Nailis et al. (2006) compararam a expressão gênica de ALS1 e ALS3 entre as células do biofilme de C. albicans formadas sobre superfície de silicone e as células em suspensão (planctônicas) e constataram um significativo aumento da expressão de ALS1 nas células do biofilme, e uma diminuição da expressão de ALS3. Por outro lado, em um estudo recente, Nobile et al. (2008) concluíram, após vários testes com mutantes als1/als1 als3/als3, que ALS3 e ALS1 são essenciais para a formação do biofilme in-vivo e a redução na expressão dessas proteínas acarreta na formação de um biofilme frágil, suas funções no biofilme são compatíveis com sua estrutura e propriedade bioquímica. Em outros estudos, o papel do produto do gene ALS1 na aderência das C. albicans às células humanas, Fu et al. (1998, 2002) constataram que o gene ALS1 codifica uma proteína de superfície celular responsável pela aderência as células endoteliais e epiteliais, e o rompimento de ambas as cópias deste gene acarretou em redução de 35% na aderência às células endoteliais, e o aumento da expressão de ALS1 elevou a aderência para 125%. Zhao et al, (2005) demostraram que a redução na expressão da proteína Als2 acarretou na redução da biomassa do biofilme, sugerindo que Als2 contribui com os estágios mais avançados do desenvolvimento do biofilme e não com o estágio da aderência. Num modelo experimental de infecção de cateter in-vivo, Als1 e Als3 também apresentaram funções redundantes; e a alta expressão de outros genes da família – ALS5, ALS6, ALS7 e ALS9 – foram capazes de substituir parcial ou completamente a ausência de ALS1 e/ou ALS3 facilitando o desenvolvimento do biofilme nesse tipo de modelo experimental, enquanto que ALS2 e ALS4 não foram capazes, e ainda, todos os genes ALS puderam ser substituídos por ALS3 ou ALS1 em modelos in-vivo e in-vitro (NOBILE et al., 2008). Em outros estudos também envolvendo cepas mutantes nocauteadas, principalmente com deleção de genes ALS, constatou-se a importância de alguns fatores transcripcionais, como Tec1 e Bcr1, bem como de outros genes codificadores, como de HWP1(hyphal wall protein) (SCHWEIZER et al., 2000; GARCIA-SANCHEZ et al., 2004; NOBILE, MITCHELL, 2005; NOBILE et al, 2006; SCHWEIZER et al., 2008; SOLL, 2008). O fato de alguns destes mutantes para ALS3 e fatores transcripcionais ainda serem capazes de formar biofilme, mesmo rudimentar e com menor espessura, sugere que essas proteínas podem não desempenhar papel fundamental durante o estágio de adesão à superfície do substrato, e sim em estágios mais � �� avançados, como por exemplo, no estágio de adesão célula-célula ou célula-hifa (BLANKENSHIP, MITCHELL, 2006). Outros autores também observaram que a expressão das Als3 e Hwp1 ocorre somente durante o estágio de hifa (STAAB, FERRER, SUNDSTROM, 1996; HOYER, PAYNE, HECHT, 1998), e que essas proteínas podem ser as mediadoras da aderência célula-hifa ou hifa-hifa (BLANKENSHIP, MITCHELL, 2006). Nobile et al. (2008) sugerem que a função de Als1 e Als3 possam ser complementares à função de Hwp1 das células vizinhas. Estes estudos também indicaram uma interessante analogia entre as adesinas de C. albicans com as mating-aglutininas de S. cerevisae, particularmente devido à similaridade estrutural de Als1 e Als3 com as �-aglutininas de S. cerevisae, proteínas estas relacionadas com a atividade sexual (mating de S. cerevisiae). Parte da estrutura da Als, incluindo a porção N-terminal, é similar à estrutura das �-aglutininas (Sag1) de S. cerevisae. As proteínas Als apresentam especificidades diferentes da Sag1, porém afinidades similares ao análogo a-aglutinina de S. cerevisae. Também foi observada analogia entre Hwp1 e a-aglutinina (AGA1 e AGA2) do S. cerevisae (Nobile et al., 2008). Essa analogia entre o tipo de resposta sexual (mating reaction) e biofilme já havia sido descrita por Daniels et al., (2006) que mostraram que o fator mating pode simular a formação do biofilme em um correspondente genético de C. albicans. Portanto, as funções complementares de Hwp1, Als1 e Als3 na formação do biofilme são análogas às funções das aglutininas sexuais durante a mating reaction. Essa associação entre biofilme e mating reaction foi também sugerida por Soll (2008), o qual ainda especula que este processo pode estar presente em outros organismos, como em Escherichia coli na qual a conjugação (mating) ocorre com freqüência 1000 vezes maior durante o biofilme do que em condições de células dispersas (GHIGO, 2001). Em resumo, esses dados sugerem que a complementaridade pode ser uma relíquia evolutiva, ou seja, uma reorganização ou aquisição de uma nova função do produto gênico de um ancestral “sexualmente mais ativo” que a C. albicans de hoje. Essa complementaridade das adesinas sugere a importância da presença de uma única espécie no biofilme, uma vez que o biofilme depende do contato intra-específico (NOBILE et al., 2008). � �� JUSTIFICATIVA O Laboratório de microbiologia Médica do Instituto de Biociências de Botucatu- UNESP possui uma importante coleção de amostras de leveduras do gênero Candida obtidas de pacientes do Hospital das Clínicas da Faculdade de Medicina de Botucatu, UNESP, com alguns isolados já previamente constatados como resistentes e outros sensíveis a alguns dos principais antifúngicos, e também produtores e não produtores de biofilme. Desse modo, o presente trabalho propôs aprofundar o estudo de caracterização destes isolados em relação a estes aspectos (sensibilidade aos antifúngicos e produção de biofilme), bem como iniciar estudos sobre a base genética deste fenótipo complexo que é a produção de biofilme, considerado fundamental na determinação da gravidade e evolução clínica das infecções causadas por estes germes. Os dados aqui obtidos poderão repercutir positivamente para o melhor entendimento e monitoramento destas infecções em nosso próprio HC, como também poderá servir de modelo e referencial comparativo para este grave problema mundial representado por essas leveduras nos ambientes hospitalares. OBJETIVOS Considerando a escassez de informações relacionada às infecções por leveduras do gênero Candida, relativa à nossa região, este estudo teve os seguintes objetivos: -caracterizar o espectro de espécies de leveduras isoladas de culturas de sangue, urina, secreção vaginal e líquido peritoneal de pacientes ambulatoriais e/ou internados no Hospital das Clínicas da Faculdade de Medicina de Botucatu; -determinar os padrões de suscetibilidade antifúngica destes isolados; -quantificar a produção de biofilme nos mesmos isolados; -caracterizar polimorfismos de tamanho da região do domínio central do gene ALS3 em isolados obtidos de hemocultura e fazer sua correlação à produção de biofilme. � �� REFERÊNCIAS BIBLIOGRÁFICAS ABI-SAID D. et al. The epidemiology of hematogenous candidiasis caused by different Candida species. Clin. Infect. Dis. v.24, p.1122-1128, 1997. APERIS G. et al. .Developments in the treatment of candidiasis: more choices and new challenges. Expert. Opin. Investig. 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Mailing address: Depto de Microbiologia e Imunologia, Instituto de Biociências, UNESP, Botucatu, São Paulo, Brasil. Abstract Background: Since opportunistic yeast infections are increasing worldwide, we carried out species identification and antifungal tests in 327 Candida isolates obtained from bloodstream infections (102 isolates), urine (85), vulvovaginal secretion (115) and peritoneal fluid (25), from a Brazilian Tertiary Clinical Hospital (UNESP School of Medicine at Botucatu, São Paulo State) from 1998 to 2005. Results: We observed 153 (46.8%) isolates of Candida albicans, 66 (20.2%) of Candida parapsilosis, 37 (11.3%) of Candida tropicalis, 29 (8.9%) of Candida glabrata, 12 (3.7%) of C. krusei and 30 (9.2%) of others species. In blood culture, C. parapsilosis was the most frequently encountered species (43.1). The resistance to antifungal agents was relatively low, while only five (3.3%) isolates of C. albicans were resistant to fluconazole, twenty one (72.4%) isolates of C. glabrata, six (50%) of C. krusei, seven (18.9%) of C. tropicalis, and two (3%) of C. parapsilosis were resistant to this drug. Resistance to itraconazole was found in eleven (7.2%) isolates of C. albicans, twenty six (89.7%) of C. glabrata, eleven (91.7%) of C. krusei, three (4.6%) of C. parapsilosis, and ten (27%) of C. tropicalis. Ketoconazole exhibited great activity against all isolates, with only two (1.3%) isolates of C. albicans being resistant. Eight amphotericin B resistant isolates were non-albicans Candida species, with six (9.1 %) being C. parapsilosis and two (10.5%) Candida spp.. Conclusions: Both the species distribution and antifungal susceptibility observed herein appear to reflect the real incidence of these opportunistic yeasts in the tertiary hospitals of Latin American countries, in which C. parapsilosis is the species most frequently encountered in bloodstream infections, while C. albicans continues to occur in an important � �� number of cases, although with a low number of resistant isolates. C. glabrata is emerging with a high number of resistant isolates, as also observed in developed countries. Background Infections caused by opportunistic pathogens, such as yeasts, are becoming important causes of morbidity and mortality in many patients, because of alterations in the immune system and invasive hospital procedures (White et al., 1998; Yang and Lo, 2001). Candidemia is commonly associated with high morbidity and mortality resulting in significant increases in the length of patients’ hospitalization and in healthcare costs (Colombo et al., 2006; Girão et al., 2008). In the past two decades, nosocomial yeast infections have increased significantly worldwide (Almirante et al., 2005; Asmundsdottir et al., 2002; Wisplinghoff et al., 2004). In the United States, yeast infection ranks as the 4th most common cause of nosocomial bloodstream infection (BSIs) (Wisplinghoff et al., 2004). In Brazil, C. albicans was the most common species isolated, followed by C. tropicalis and C. parapsilosis. In addition, the study revealed that antifungal resistance was rare (Colombo et al., 2006). There has been an important shift in the species causing nosocomial candidemia, with the emergence of non- albicans species particularly more resistant to antifungal drugs (Abi-Said et al., 1997; Clark & Hajjed, 2002, Trick et al., 2002; Snydman, 2003; Sobel, 2006). Several antifungal drugs have been used to control such infections, and as a result of broad prophylactic usages and long-term treatments with those drugs, the prevalence of drug resistance has become an important issue in various yeast infections, thus profoundly affecting human health (Marr et al., 2001; Pfaller et al., 2003; Yang et al., 2004). Candida species have various degrees of susceptibility to the frequently used antifungal drugs. Candida krusei is intrinsically resistant to fluconazole, and Candida glabrata is less susceptible or has higher MICs than other Candida species (Akova et al., 1991; Orozco et al., 1998; Yang et al., 2004). In the present work, we present data on species frequency and antifungal susceptibility of Candida isolates obtained in a Brazilian public tertiary hospital. � �� Methods Origin of the isolates: All the yeast cultures were obtained from patients of the Clinical Hospital of the UNESP School of Medicine, Botucatu, São Paulo State, between 1998 and 2005. The isolates were stored in vial tubes containing Brain Heart Infusion plus 10% glycerol, in a freezer at -80°C. At the moment of the study each isolate was cultured on Sabouraud Dextrose Agar plates at 35ºC. Species identification: Species identification was based on the colony morphology on Chromogenic Agar (CHROmagar Candida, Difco), microscopy features on Corn-meal Agar slide culture, as well as the assimilation and fermentation tests, according to Kurtzman & Fell (1998). Isolates that not fit any recognized taxon were considered Candida spp. Susceptibility tests: Minimal inhibitory concentrations (MIC) of fluconazole (Pfizer, São Paulo, Brazil), itraconazole (Janssen, Beerse, Belgium), ketoconazole (Janssen, Beerse, Belgium) and amphotericin B (Sigma, St. Louis, MO, USA) were determined by broth microdiluition according to the Clinical and Laboratory Standard Institute (CLSI) document guidelines for the susceptibility testing of yeasts (CLSI, M27-A2, 2002). C. parapsilosis ATCC 22019 and C. krusei 6258 were used for quality control on each test run. For the azole drugs, the MIC was defined as the lowest concentration corresponding to 50% inhibition compared with growth in the drug-free control well (CLSI, M27-A2, 2002; Espinell-Ingroff et al., 2005). For amphotericin B, the MIC was considered the lowest concentration showing growth inhibition (CLSI, M27-A2, 2002). For susceptibility to fluconazole, isolates with MIC �64 �g/mL were considered resistant, whereas those with MIC �8 �g/mL were susceptible. For susceptibility to itraconazole, isolates with MIC �1 �g/mL were defined as resistant, whereas those with MIC �0.125 �g/mL were susceptible. Isolates with MICs falling between 16 and 32 �g/mL in relation to fluconazole, 0.25–0.5 �g/mL to itraconazole were defined as dose- dependent susceptibility. With regard to ketoconazole, isolates with MIC >16 �g/mL were defined as resistant, whereas those with MIC � 0.03 �g/mL were susceptible while those with MIC between 0.03-16 �g/mL presented dose-dependent susceptibility, according to the E-test® recommendation. For susceptibility to amphotericin B, isolates with MIC �2 �g/mL were considered resistant, and those with MIC �1 �g/mL were susceptible (Nguyen et al., 1998, Yang et al., 2008). Where ten or more species were tested, the MIC50 and the MIC90 were calculated. � �� Results Species identification: In a total of 327 yeast cultures, 153 (46.8%) were isolates of C. albicans, sixty six (20.2%) C. parapsilosis, thirty seven (11.3%) C. tropicalis, twenty nine (8.9%) C. glabrata, twelve (3.7%) C. krusei, nine (2.8%) C. guilliermondii, one (0.3%) C. lusitaniae, one (0.3%) C. pelliculosa and nineteen (5.8%) Candida spp.(Table 1). With regard to the clinical materials, while C. albicans continued as the most frequent species in urine (34.1%) and in vulvovaginal secretions (80.9%), C. parapsilosis was the most frequent in blood (43.1%) and in peritoneal fluid (40.0%). C. tropicalis, the third most frequent species, occurred mainly in urine and vulvovaginal secretions (Table 1). Susceptibility tests: Susceptibility tests for fluconazole, itraconazole, ketoconazole and amphotericin B were performed on 327 isolates of Candida species. Table 2 shows the MIC ranges delimiting inhibition of isolates at proportions of 50% and 90%, as well as the percentages of isolates resistant to the four antifungal drugs tested. Overall, the resistance to antifungal agents was relatively low, especially for C. albicans. Among the 327 evaluated isolates, forty-one (12.5%) were resistant to fluconazole, fifty-eight (17.7%) to itraconazole, two (0.6%) to ketoconazole and eight (2.4%) to amphotericin B. Fluconazole exhibited the greatest activity against C. albicans with resistance shown by only five (3.3%) isolates. Twenty-one (72.4%) isolates of C. glabrata, six (50%) of C. krusei, seven (18.9%) of C. tropicalis, and only two (3%) of C. parapsilosis were resistant to fluconazole. The fluconazole MIC50 and MIC90 for C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and Candida spp. were 0.5 and 8, 64 and 64, 32 and >64, 2 and 8, 4 and >64, and 2 and 16 �g/mL, respectively. Resistance to itraconazole was found in eleven (7.2%) isolates of C. albicans, twenty- six (89.7%) of C. glabrata, eleven (91.7%) of C. krusei, three (4.6%) of C. parapsilosis, and ten (27%) of C. tropicalis. The MIC50 and MIC90 for itraconazole against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and Candida spp. were 0.03 and 0.12, 2 and >16, 2 and 2, 0.03 and 0.12, 0.12 and >16, and 0.03 and 0.12 �g/mL, respectively. Ketoconazole exhibited great activity against all isolates, with only two (1.3%) isolates of C. albicans resistant. The MIC50 and MIC90 for ketoconazole against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and Candida spp. were 0.03 and 1, 2 and 4, 2 and 4, 0.06 and 0.12, 1 and 8, and 0.06 and 0.25 �g/mL, respectively. � �� The eight amphotericin B-resistant isolates were non-albicans Candida species, consisted of six (9.1%) C. parapsilosis and two (10.5%) Candida spp.. The MIC50 and MIC90 for amphotericin B against all isolates were 1 �g/mL, except for C. krusei (0.5�g/mL), C. parapsilosis (0.12 and 1 �g/mL) and Candida spp (0.5 and 0.25 �g/mL) (Table 2). All isolates of C. guilliermondii (nine), C. lusitaniae (one) and C. pelliculosa (one) were susceptible to amphotericin B and to the three azoles, except one isolate of C. guilliermondii that was resistant to fluconazole and intraconazole. Discussion The frequency of infections caused by yeasts, especially Candida spp., has increased dramatically worldwide in recent years (Pfaller & Diekema., 2002; Hajjeh et al., 2004). Although C. albicans remains as the most frequent species, several other Candida species are emerging, and in some casuistic cases non-albicans ones are the most frequent (Colombo et al., 2006; Caggiano, et al., 2007; Celebi et al., 2007; Shivaprakasha et al., 2007; Costa-de-Oliveira et al., 2008; González et al., 2008; Kersun et al., 2008). In the present study, carried out in a Brazilian public tertiary hospital, the frequency of non-albicans species represented 53.2% of all Candida isolates. C. parapsilosis was the most frequent non-albicans species recovered, occurring in 20.2% of all isolates, followed by C. tropicalis (11.3%) and C. glabrata (8.9%). Other studies have also indicated an important occurrence of C. parapsilosis, in Brazil, mainly associated with bloodstream infections (Colombo et al., 1999; Passos et al., 2007) and, in the present case, also found in peritoneal fluid. Ruiz et al. (2005) and Medrano et al. (2006) showed that C. parapsilosis was the species most frequently isolated from bloodstream infections, both in the southeast and northeast regions of Brazil. The real reasons why C. parapsilosis occurs more frequently in Latin American countries is not completely understood. C. parapsilosis is considered a commensal of human skin since it has been isolated from the hands of health workers (Asbeck et al., 2007; Trofa, et al., 2008), which have been identified as the major vectors in the infection acquisition (Trofa et al., 2008). Some important virulence factors have been observed in C. parapsilosis, such as adherence to host cells, biofilm formation and production of hydrolytic enzymes (Branchini et al., 1994; Trofa et al., 2008). Furthermore, this species presents selective growth capability in hyperalimentation solutions and an affinity for intravascular devices and prosthetic materials (Clark, et al., 2004; Trofa et al., 2008). The lead occurrence of C. parapsilosis in the peritoneal fluid in our casuistic case � � also comes as no surprise, and this fact has also been observed in different countries in patients receiving peritoneal dialysis (Wang et al., 2000; Manzano-Gayoso et al., 2003). The adoption of good infection control practices, with adequate asepses of health workers’ hands and medical devices, especially in catheters, may substantially minimize infection by C. parapsilosis. While in developed countries C. glabrata has been considered the most frequent among non-albicans species, in the present study C. glabrata was only the fourth most frequent, occurring in blood, urine and vulvovaginal secretions. Under our casuistic, C. krusei was isolated only from vulvovaginal secretions, at a frequency similar to those observed in other distant areas, such as in Asia (Chong et al. 2007). Invasive infections by C. krusei in Brazil appear to be less frequent than in developed countries, as already observed in other local studies (Matsumoto et al., 2002; Antunes, et al., 2004; Colombo et al., 2006). In a worldwide surveillance study, Pfaller et al., (2008) indicated that C. krusei represents 3.3% of all Candida spp. isolated in Europe and North America and 1.7% in Latin America. C. guilliermondii, considered a normal component of human skin and mucosal flora (Mok and Barreto Silva, 1984), is rarely associated with invasive infections like candidemia and peritonitis (Pasqualotto et al., 2006). In the present work, C. guilliermondii was isolated from blood, peritoneal fluid and urine. Recent reports have shown cases of candidemia by C. guilliermondii in several countries including Brazil (Colombo et al., 2006; Caggiano et al., 2007; Lee et al., 2007; Odds et al., 2007; Passos et al., 2007; Gonzáles et al., 2008). Other reports have also found few cases of candidemia by C. lusitaniae and C. pelliculosa in Brazil (Colombo et al., 2006; Passos et al., 2007; França et al., 2008) and other countries (Caggiano et al., 2007; Odds et al., 2007; Kersun et al., 2008). Paradoxically, the increased attention, monitoring and use of antifungal drugs to treat yeast infections have coincided with both the emergence of non-albicans species and an augmented number of resistant strains. Fluconazole has been considered the antifungal of choice for the empirical treatment of suspected infection caused by any species of Candida. However, several studies have clearly indicated the necessity of correct species identification, since they may differ substantially in relation to drug susceptibility (Mensa et al., 2008). In the present study, most of the isolates were susceptible to the antifungal drugs tested. Fluconazole and itracoanazole resistance were observed in few isolates of C. albicans, C. parapsilosis, C. tropicalis and C. guilliermondii. Similar to other studies, the percentage of isolates resistant to fluconazole was smaller than to itraconazole (Dóczi et al., 2002; Cheng et al., 2004; Laverdiere et al., 2007; González et al., 2008). As expected, C. krusei and C. glabrata isolates � � displayed high resistance to fluconazole and itraconazole, since they are considered intrinsically resistant to these drugs (Mensa et al., 2008). Bloodstream infections by C. krusei and C. glabrata are associated with high mortality, because of their poor response to conventional therapy (Costa-de-Oliveira et al., 2008; González et al., 2008). Although the incidences of these two species under our case casuistic have been relatively low, it is important to be vigilant in monitoring these agents, mainly in the patients receiving antifungal azole drugs. The high susceptibility to ketoconazole shown by all isolates in our casuistic cases might also explained by the fact that this drug has not been prescribed in our hospital in recent years (data provided by the local control infection committee – Mondelli, personal communication) In our study, resistance to amphotericin B was observed only in non-albicans species, mainly in C. parapsilosis, the most frequent non-albicans species. Although the majority of pertinent studies report a lack of amphotericin B-resistant isolates (Colombo et al., 2006; Odds et al., 2007; Arendrup et al., 2008; Chen et al., 2008; Kersun et al., 2008), another Brazilian study also found resistance by C. parapsilosis to amphotericin B (Passos et al., 2007). 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Clin Infect Dis 2002, 35:627-630. Trofa D,D Gácser A, Nosanchuk JD: Candida parapsilosis, an emerging fungal pathogen Clin Microbiol Rev 2008, 21:606-625. Wang AY, Yu AW, Li PK, Lam PK, Leung CB, Lai KN, Lui SF: Factors predicting outcome of fungal peritonitis in peritoneal dialysis: analysis of a 9-year experience of fungal peritonitis in a single center. Am J Kidney Dis 2000, 36:1183-1192. White TC, Marr KA, Bowden RA: Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998, 11:382-402. � �� Wisplinghoff H, Bischoff T, Tallent SM, Seifert H,Wenzel RP, Edmond MB: Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004, 39:309-317. Yang YL, Ho YA, Cheng HH, Ho M, Lo HJ: Susceptibilities of Candida species to amphotericin B and fluconazole: the emergence of fluconazole resistance in Candida tropicalis. Infect Control Hosp Epidemiol 2004, 25:60–64. Yang YL, Lo HJ: Mechanisms of antifungal agent resistance. J Microbiol Immunol Infect 2001, 34:79-86. Yang YL, Wang AH, Wang CW, Cheng WT, Li SY, Lo HJ, TSARY Hospitals: Susceptibilities to amphotericin B and fluconazole of Candida species in Taiwan Surveillance of Antimicrobial Resistance of Yeasts – 2006. Diag Microbiol Infect Dis 2008, 61:175-80. � �� Table 1 – Distribution frequency of Candida species obtained from different clinical materials at the Brazilian Tertiary Hospital (Clinical Hospital of the UNESP School of Medicine, Botucatu, São Paulo State), from 1998 to 2005. Species Blood Urine Vulvov. Per. fluid Total identification % (n) % (n) % (n) % (n) % (n) C. albicans 22.5 (23) 34.1 (29) 80.9 (93) 32.0 (8) 46.8 (153) All non-C. albicans species 77.5 (79) 65.9 (56) 19.1 (22) 68.0 (17) 53.2 (174) C. glabrata 4.9 (5) 23.5 (20) 3.6 (4) - 8.9 (29) C. guilliermondii 5.9 (6) 1.2 (1) - 8.0 (2) 2.8 (9) C. lusitaniae 1.0 (1) - - - 0.3 (1) C. parapsilosis 43.1 (44) 8.2 (7) 4.5 (5) 40.0 (10) 20.2 (66) C. pelliculosa 1.0 (1) - - - 0.3 (1) C. tropicalis 2.9 (3) 32.9 (28) 0.9 (1) 20.0 (5) 11.3 (37) C. krusei - - 10.4 (12) - 3.7 (12) Candida spp. 18.6 (19) - - - 5.8 (19) Total 102 85 115 25 327 � �� Table 2. In vitro activity of antifungal agents against Candida spp. isolates from different clinical materials at the Brazilian Tertiary Hospital (Clinical Hospital of the UNESP School of Medicine, Botucatu, São Paulo State), from 1998 to 2005. Isolates (n) Drugs MIC (μg/ml) Range MIC50 MIC90 R n(%) C. albicans (153) FLU 0.03->64 0.5 8 5 (3.3) ITR 0.03->16 0.03 0.12 11 (7.2) KET 0.03->16 0.03 1 2 (1.3) AMB 0.06-1 1 1 0 C. glabrata (29) FLU 2->64 64 64 21 (72.4) ITR 0.06->16 2 >16 26 (89.7) KET 0.03-16 2 4 0 AMB 0.5-1 1 1 0 C. krusei (12) FLU 32->64 32 >64 6 (50) ITR 0.5-4 2 2 11 (91.7) KET 1-4 2 4 0 AMB 0.5-1 0.5 0.5 0 C. parapsilosis (66) FLU 0.12-64 2 8 2 (3) ITR 0.03->16 0.03 0.12 3 (4.6) KET 0.03-4 0.06 0.12 0 AMB 0.25-2 1 1 6 (9.1) C. tropicalis (37) FLU 0.12->64 4 >64 7 (18,9) ITR 0.03->16 0.12 >16 10 (27) KET 3-16 0.12 8 0 AMB 0.5-1 1 1 0 Candida spp. (19) FLU 0.25-16 2 16 0 ITR 0.03-0.25 0.03 0.12 0 KET 0.03-0.25 0.06 0.25 0 AMB 0.5-2 0.5 2 2 (10.5) FLU: fluconazole, ITR: itraconazole, KET: ketoconazole, AMB: amphotericin B � � Biofilm production and ALS3 central domain polymorphism in Candida species from different clinical sources. Ariane Bruder-Nascimento; Maria Fátima Sugizaki; Carlos Henrique Camargo; Eduardo Bagagli* *Corresponding author. Mailing address: Depto de Microbiologia e Imunologia, Instituto de Biociências, UNESP, Botucatu, São Paulo, Brasil. Abstract Biofilm production was quantified in 327 Candida species isolated from the bloodstream, urine, vaginal secretion and peritoneal fluid obtained from a Brazilian tertiary public hospital (Clinical Hospital of UNESP, School of Medicine, Botucatu, São Paulo State). Of the 198 total biofilm-positive isolates, 72 and 126 were considered low and high biofilm producers, respectively. Biofilm production by C. albicans isolates was significantly lower than that by non-albicans isolates, and among the biofilm-positive isolates, the non-albicans isolates were classified mainly as high-biofilm producers, while C. albicans isolates presented low biofilm production. Such production was most frequently observed in C. tropicalis isolates, followed by C. parapsilosis, C. glabrata, and C. albicans. Among biofilm-positive isolates, the highest production intensity was observed in C. tropicalis isolates. Biofilm production was more frequent among bloodstream isolates than any other clinical source. 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, which must have originated from systemic infection, reflecting the close association between biofilm production and virulence. The ALS3 central domain polymorphism was also evaluated in the bloodstream isolates, including C. albicans and non-albicans species, both biofilm-positive and -negative producers. The numbers of tandem-repeat copies were divergent among the isolates, which presented homozygosity and heterozygosity, and varied from 7 to 14 copies. The numbers of tandem-repeat copies per allele appear to be unassociated with biofilm production while the respective mean numbers of copies, in biofilm-negative and biofilm-positive isolates, were 11.6 ± 1.4 and 10.7 ± 1.7. � � Introduction Candida species are human commensals that can cause both superficial and systemic disease, mainly in immunocompromised individuals (Kojic & Darouiche, 2004). These organisms have emerged as important agents of opportunistic infections worldwide, primarily in immunocompromised persons (Richardson, 2005; Aperis et al.,2006). Although Candida albicans is considered the most common fungal pathogen, an increased number of non- albicans Candida species infections have been described (Redding, 2001; Krcmery & Barnes., 2002). Candida species can colonize human tissues and medical devices, such as central venous catheters, prosthetic heart valves and other devices, resulting in biofilm formation and biofilm-related infections (Douglas, 2003; Andes et al., 2004; Kojic & Darouiche, 2004). Biofilms are microbial communities of surface-attached cells embedded in a self-produced extracellular polymeric matrix (Donlan and Costerton, 2002). They can cause significant problems in many areas, mainly in medical settings as persistent and recurrent device-related infections (Flemming, 2002; Fux et al., 2005; Kumar & Anand, 1998). Biofilms are more resistant than planktonic cells, and in most cases, antifungal therapy is not effective (Douglas, 2003; Kumamoto, 2002). The implanted device that is most commonly infected is the central venous catheter, which is used to administer fluids and nutrients as well as cytotoxic drugs. Infections can arise at any time during the use of this type of catheter. (Goldmann &. Pier, 1993; Douglas, 2002). However, endogenous infections also can occur if Candida spp. colonizing the gastrointestinal tract as commensals are able to penetrate the intestinal mucosa and invade the bloodstream, after which circulating yeasts can contact the catheter tip internally (Goldmann &. Pier, 1993). But non-device-related infections can also involve biofilms, for example in Candida endocarditis and Candida vaginitis (Donlan & Costerton, 2002; Douglas, 2002). Biofilm formation in Candida spp. is a complex process involving multiple regulatory mechanisms (Nobile & Mitchell, 2006) and once established, Candida biofilms serve as a persistent reservoir of infection and, in addition, offer greater resistance to antifungal agents compared to planktonic phase yeasts (Chandra et al., 2001a,b; Samaranayake, et al., 2005; Parahitiyawa et al., 2006). Several different biofilm in-vitro systems have been developed to study and quantify biofilm, including yeast development on intravascular catheter discs, acrylic discs, cylindrical cellulose filters, microtiter plates and others (Douglas, 2002; McLean et al., 2004). � �� Crystal violet staining, a basic dye that binds to negatively charged surface molecules and polysaccharides in the extracellular matrix, is commonly utilized to quantify biofilms formed by a broad range of microorganisms, including yeasts (Christensen et al., 1985; Jin et al., 2003; Li et al., 2003; Peeters et al., 2008). Besides phenotypical assays to study biofilm formation in Candida species, some genotypical techniques have been used to characterize this phenomenon. The Als (agglutinin- like sequence) proteins have long been considered excellent candidates for biofilm adhesions (Green et al., 2004, Hoyer et al., 1998; Zhao et al., 2006). Eight ALS genes (ALS1 to ALS7 and ALS9) encode large, cell surface glycoproteins, some of which promote adhesion to host surfaces (Fu, et al., 2002; Gaur et al., 1997; Hoyer, 2001, Zhao et al., 2003; Zhao et al., 2004). ALS genes have three general domains: the 5’domain, conserved, with approximately 1300-pb; followed by a central domain consisting entirely of tandem repeats of a 108-pb sequence, that are somewhat variable, and the 3’ domain, which is least conserved in length and sequence (Hoyer, 2001; Hoyer et al., 2008). Although they share a similar three-domain structure, sequence differences among the Als proteins can be large, suggesting that the proteins may present different functions (Hoyer, 2001). Much of the allelic variation in ALS genes occurs within the tandem repeat domain and is manifested as differing numbers of the 108-pb tandem repeats in ALS alleles. It has recently been suggested that ALS3 is one of the most important genes associated with C. albicans biofilm production (Zhao et al., 2006, Hoyer, 2001; Hoyer et al., 1998). The aim of the present study was to quantify biofilm production in a collection of different Candida species, isolated from the bloodstream and other different clinical sources, as well as to detect the polymorphisms in the ALS3 tandem repeat domain and their possible correlation with the biofilm production profiles. Materials and Methods Microorganisms: A total of 327 Candida species isolates recovered from clinical specimens as part of routine diagnostic procedures, stored in vial tubes containing Brain Heart Infusion plus 10% glycerol, frozen at -80°C, were re-cultured and tested for biofilm production. The isolates were obtained from patients from the Clinical Hospital of UNESP (State University of Sao Paulo) School of Medicine, Botucatu, São Paulo State (HC/UNESP), between 1998 and 2005. The isolates were obtained from the bloodstream (102), urine (85), � �� vulvovaginal secretion (115) and peritoneal fluid (25). The Candida species studied included 153 C. albicans, 66 C. parapsilosis, , 37 C. tropicalis, 29 C. glabrata, 12 C. krusei, 9 C. guilliermondii, 1 C. pelliculosa, 1 C. lusitaniae and 19 Candida spp. (species belonging to the genus Candida that did not fit any recognized taxon). The identification of Candida species was conducted by chlamydospore formation, sugar assimilation and fermentation patterns as well as chromogenic agar (CHROMagar Candida, Difco). The species distribution in different materials is summarized in Table 1. Biofilm formation assay: Tests for biofilm quantification were performed according to Li et al. (2003) and Jin et al. (2003), with few modifications. Growth conditions: Briefly, to prepare inoculum, all isolates were first streaked onto Yeast-extract peptone dextrose agar (YEPD) and incubated at 37 °C for 48 h. For each isolate, a large loop of actively growing cells was transferred to sterile Yeast Nitrogen Base (YNB) broth (Difco) containing 0.9 % D- glucose. After incubation at 37 °C for 24h, the yeast cells were centrifuged and washed twice with 0.5 mL PBS (0.14 M NaCl, 2.7 mM KCl, 8.5 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) by vortexing and centrifuging at 5000g for 5 min. The washed cells were then re-suspended in 1mL YNB broth and the concentration was adjusted to 107 cells/mL, according to 0.5 McFarland scale. Biofilm formation: For each isolate, 100 μL of the suspension was inoculated into individual wells of polystyrene 96-well plates (TPP). Four repetitions were performed for each isolate. YNB broth containing no inoculum was used as negative control. The plates were incubated at 37 °C for 90 min (adhesion period). Supernatant including planktonic cells and liquid medium was then discarded and wells were gently washed twice with PBS to eliminate any non-adherent cells. For biofilm growth, 100 μL of fresh YNB broth was then added to each well. The plates were incubated at 37 °C for 48h. After biofilm formation and growth, planktonic cells were discarded through three rounds of washing with 200 μL sterile PBS buffer, and the plates dried at room temperature for 45min. For staining with Crystal Violet (CV), 150μL of 0.4 % CV, diluted in water, was added to each well, and after 45 min at room temperature, all the supernatant was discarded before adding 150 μL of 95 % ethanol and maintained for 45 min, to dissolve and/or elute the dye from the biofilm cells. Next, 100 μL of each well was transferred to a new 96-well microplate and the absorbance determined using a microplate reader at 540nm filter (MultisKan EX, Labsystems). The wells containing only YNB broth with no yeasts were used as negative controls. The absorbance values were converted into transmittance percentages (%T). The %T values for each test was subtracted from the %T for the reagent blank to obtain a measure of light blocked when passing through � � the wells (%Tbloc), and the biofilm production scored as either negative (%Tbloc <10), positive 1+ (%Tbloc 10 to 20), positive 2+ (%Tbloc 20 to 35), positive 3+ (%Tbloc 35 to 50) or positive 4+ (%Tbloc �50), and the positives further categorized as low-biofilm (1+) or high-biofilm producers (2+, 3+, or 4+), according to Tumbarello et al. 2007. ALS3 characterization: The gene ALS3 was studied in all C. albicans isolates, biofilm producers and non-producers, obtained from bloodstream culture (n=23), as well as in 7 isolates of C. parapsilosis, 3 of C. tropicalis, 1 of C. guilliermondii 1 of C. glabrata and 4 of Candida spp., all also isolated from the bloodstream. DNA extraction: The DNA was extracted according to McCoullogh et al. (2000) with few modifications. Yeasts were grown for 24h on Sabouraud dextose agar at 37°C. Colonies were suspended in 1 mL of 1 M sorbitol and 125 mM of EDTA. The suspension was centrifuged (10 min at 13000 g), the supernatant was discarded, and the pellet was resuspended in 0.5 mL of lysing solution (1 M Tris-HCl, pH 8.0, with 250 mM of EDTA and 5% SDS) plus 10 μL of proteinase K (Invitrogen) and incubated for 1 h at 65°C. Next, 500 μL 5 M potassium acetate was added, incubated on ice for 2 h and then centrifuged (10 min at 13,000 g). The supernatant was transferred to an Eppendorf tube containing 1 mL of 100% ethanol. This was mixed by inversion and centrifuged (10 min at 13,000 g and 4°C). The supernatant was discarded, the pellet was washed with 500 μL of cooled 70% ethanol and centrifuged (10 min at 13,000 g and 4°C). The supernatant was discarded and the pellet was resuspended in 0.5 mL of sterilized MiliQ water. PCR conditions: The size of the tandem repeat domain in each ALS allele was determined by PCR using two independent primer pairs as described by Oh et al. (2005). Two primer pairs provided an additional control for the accuracy of the results. Each primer pair contained one that annealed 5' and another 3' of the tandem repeat domain. The first primer pair was ALS3GenoF (5'-ACC TTA CCA TTC GAT CCT AAC C-3') and ALS3GenoR (5'-GAT GGG GAT TGT GAA GTG G-3'). The second primer pair was ALS3GenoF2 (5'-CCA CAA CAC ATA CTA ATC CAA CTG A-3') and ALS3GenoR2 (5'-TGT AGA CCA CAA AGT TGT ATG GTT G-3'). Taq polymerase (Invitrogen) was used with both primer pairs. Reactions with the first primer pair (ALS3GenoF and ALS3GenoR) used Invitrogen Taq polymerase buffer with 1 mM MgCl2. Reactions were heated for 5 min at 94 °C followed by 35 cycles of 94 °C (30 s), 57 °C (30 s) and 72 °C (3 min). A final 72 °C (7 min) extension completed the reaction. The second primer pair (ALS3GenoF2 and ALS3GenoR2) was used under similar conditions except for a difference in buffer (10 mM Tris/HCl, pH 8·8, 25 mM KCl, 1·5 mM MgCl2) and annealing temperature (65 °C). When the first pair of primers provides no clear amplification, � �� the second pair was used. PCR products were separated on 0.7 % agarose (TBE) gels stained with ethidium bromide. The gels were analyzed in the equipment AlphaImagerR EC that captures the digital image whereas the sizes of the amplicons were determined by the software AlphaEaseR FC. To estimate, for each isolate, the numbers of motifs present in the tandem repeats in the ALS3 gene, the primers positions were aligned with the deposited genomic sequences of strain SC5314 DNA (GenBank Accession No. AY223552.1), large and small alleles that present twelve and nine motifs, using Mega software. The numbers of motifs for each isolate evaluated were then calculated, considering 108 bp the mean size for each motif. The amplicon of one homozygous isolate was also purified by the commercial kit GFX PCR DNA and Gel Band (GE, Healthcare), sequenced using the DYEnamic ET Dye Terminator Kit (with Thermo Sequenase™ II DNA Polimerase) in a MegaBACE 1000 DNA Analysis System, and the chromatogram visualized by the Chromas program. The consensus sequence was sent to blastn for comparison with the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST). Statistical analysis. Chi-square ana