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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
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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
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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.
...e
nfim, 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.
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Species distribution and susceptibility profile of Candida species in a Brazilian Public
Tertiary Hospital
Authors: Ariane Bruder-Nascimento; Maria Fátima Sugizaki; Terue Sadatsune; Augusto Cezar
Montelli; Alessandro Lia Mondelli; Eduardo Bagagli*
*Corresponding author. 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 MIC
50
and the MIC
90
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 MIC
50
and MIC
90
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 MIC
50
and MIC
90
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 MIC
50
and MIC
90
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 MIC
50
and MIC
90
for amphotericin B against all isolates were 1 g/mL, except for C. krusei (0.5g/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).
Amphotericin B is particularly used in several Brazilian public tertiary hospitals in the
treatment of systemic mycosis, in which the patients remain hospitalized for long periods of
treatments, as in our hospital for paracoccidiodomycosis (Dillon et al., 1986). The possible
effect of this drug against selectively resistant Candida species should not be excluded and
merits proper evaluation.
Acknowledgements
We thank Carlos Henrique Camargo for the lab assistance and staff of the Laboratory of
Medical Microbiology of the Clinical Hospital of the UNESP School of Medicine, Botucatu,
São Paulo State, for their help in providing the isolates.
This work was supported by Fapesp (grant n º 2007/01946-4).
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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. para
psilosis
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 MIC
50
MIC
90
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 (7
2.4)
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 Na
2
HPO
4
, 1.8 mM
KH
2
PO
4
, 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 10
7
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 (%T
bloc
), and the biofilm production scored
as either negative (%T
bloc
<10), positive
1+ (%T
bloc
10 to 20), positive 2+ (%T
bloc
20 to 35), positive 3+ (%T
bloc
35 to 50) or positive 4+
(%T
bloc
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 MgCl
2
. 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 MgCl
2
) 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 AlphaImager
R
EC that
captures the digital image whereas the sizes of the amplicons were determined by the software
AlphaEase
R
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 analysis was used to compare biofilm positivity among
different Candida species or among isolates recovered from different sources. The
Kolmogorov-Smirnov test was used to compare the numbers of tandem repeat copies between
biofilm-positive and biofilm-negative isolates. Differences between groups were considered to
be significant for P < 0.05.
Results
Biofilm production: Biofilm production for 327 isolates of Candida species obtained
from different sources is summarized in Figure 1 and Table 2. A total of 198 of 327 (60.6%)
Candida species isolates were biofilm-positive. Of these, 72 and 126 isolates were low and
high biofilm producers, respectively. Biofilm production by C. albicans isolates (43.1%, 66 of
153) was significantly lower than that by non-albicans isolates (75.8%, 132 of 174; P < 0.001),
and among the biofilm-positive isolates, the non-albicans isolates were classified mainly as
high-biofilm producers, with C. albicans isolates defined as low-biofilm producers (P<0.001).
Considering the species separately,
biofilm
production was most frequently observed in
C. tropicalis isolates (94.6%, 35 of 37, P < 0.001), followed by C. parapsilosis (80.3%, 53 of
66), C. glabrata (44.8%, 13 of 29), and C. albicans (43.1%, 66 of 153).
Among biofilm-
positive isolates, the highest biofilm production intensity (%T
bloc
> 35) was observed in C.
tropicalis isolates (81.1%, 30 of 37, P < 0.001).
In relation to the sources, biofilm production was positive in 79.4% (81 of 102) of the
isolates obtained
from the bloodstream, 63.5% (54 of 85) from urine, 37.4% (43 of 115) from
vaginal secretion and 80% (20 of 25) from peritoneal fluid. Biofilm production among
bloodstream isolates was more frequent than any other source (P < 0.001), except peritoneal
fluid isolates, which are also prolific biofilm producers. Biofilm production was most frequent
in non-albicans isolates (P < 0.001) in all sources, also with the exception of peritoneal fluid
isolates (P > 0.05). In urine, the isolates displayed a peculiar distribution by presenting two
distinct peaks, one containing biofilm-negative isolates (36.5%) and the other containing
isolates (37.6%) with intense biofilm production (%T
bloc
> 35).
ALS3 characterization: Polymerase chain reaction was performed on a total of 39
isolates, including 23 C. albicans and 16 non- albicans species. In 19 of 23 C. albicans
isolates, the expected fragments of the ALS3 gene were amplified by using the first or the
second pair of primers (Figure 2). In the 16 non-albicans evaluated, the PCR products were not
amplified with both pairs of primers.
The consensus sequence of a fragment containing approximately 700 bp in the Blast
analysis presented 93% identity with the C. albicans large allele ALS3 gene (GenBank
Accession No. AY223552.1; E value 0.0).
The numbers of copies of the central tandem repeat domain were divergent among the
isolates, which presented 74% homozygosity and 26% heterozygosity. Alleles in the examined
isolates encoded between 7 and 14 copies of tandemly repeated 108-bp sequence (Table 3).
The mean numbers of tandem repeat copies per allele in biofilm-negative and biofilm-positive
isolates were 11.6 ± 1.4 and 10.7 ± 1.7, respectively. The difference in the number of tandem-
repeat copies between biofilm-positive and biofilm-negative isolates was not significant (P >
0.05).
Discussion
Biofilm production has been considered an important virulence factor among Candida
species (Ramage et al., 2005). A broad range of methods has been employed to evaluate this
phenomenon in yeasts, mainly in Candida species (Peeters et al., 2008). In the present study,
the biofilm production was quantified in 327 clinical isolates of Candida species by the crystal
violet assay, a widely used method to quantify biofilm production in several microorganisms,
including yeasts (Christensen et al. 1985; Stepanovic et al., 2000; Li et al., 2003; Peeters et al.,
2008). Although this method is not able to differentiate between living and dead cells (Pitts et
al., 2003; Peeters et al., 2008), the results obtained by the crystal violet assay have correlated
highly with other assays that differentiate between living and dead cells, such as the 2,3-bis (2-
methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)
assay (Jin et al., 2003; Peeters et al., 2008). The data obtained herein clearly confirm that
different Candida species have different abilities to produce biofilm in-vitro and that non-
albicans species were the highest biofilm producers (Shin et al., 2002; Tumbarello et al.,
2007). Among biofilm producers, C. tropicalis showed the highest intensity of biofilm
production. In other studies involving similar numbers of Candida species isolates, C.
tropicalis was the species with the highest percentages of biofilm positivity, while C. albicans,
ranked third or forth among the biofilm-positive isolates (Shin et al., 2002; Tumbarello et al.,
2007).
We also examined the associations between the source and the biofilm positivity of
isolates. Isolates obtained from the bloodstream and peritoneal fluid typically are prolific
biofilm producers. Considering that isolates from invasive infections produce more biofilm
than those from non-invasive infections (Kuhn et al., 2002), we suggest that some isolates
obtained from urine presenting intense biofilm production may have originated from systemic
candidiasis and not from urinary tract infections.
Although important advances have been achieved in outlining the genetic basis of
biofilm production, the subject is far from being completely understood. After the C. albicans
genome was sequenced (Jones et al., 2004), the biofilm production by this yeast was better
comprehended, although biofilm formation in non-albicans species remains poorly understood
(Weber et al., 2008). Several genes are involved in the biofilm formation by Candida spp.
(López-Ribot, 2005; Ramage et al., 2005; Nobile & Mitchell 2006). It was demonstrated that
the expression of several ALS genes is upregulated during biofilm formation; furthermore, the
Als proteins have long been considered excellent candidates for biofilm adhesins (Green et al.,
2004; Blakenship & Mitchell, 2006). The present study analyzed the association between
biofilm production and polymorphisms in the ALS3 central domain. Results from previous
studies already showed that ALS3 gene expression is altered in C. albicans sessile cells
compared to planktonic cells (García-Sánchez et al., 2004; Nailis et al., 2006; Chandra et al.,
2001a,b). Maximal ALS3 expression is associated with formation of germ tubes and hyphae
(Hoyer et al., 1998) and a recent work demonstrates an overexpression of ALS3 in the initial
stages of biofilm formation (Nailis et al., 2009). In our results, the association between the
number of tandem-repeat copies per allele and biofilm production was not significant.
However, biofilm-negative isolates showed the majority of alleles with 13 tandem-repeat
copies, while the majority of alleles in biofilm-positive isolates showed 10 or 11 copies. The
fact that both primer pairs tested herein had been previously designed to study C. albicans
certainly explains why none of the studied non-albicans species produced PCR products, due
to the different sequences at the annealing sites, between C. albicans and non-albicans species.
The absence of amplification in four C. albicans isolates suggests the presence of sequence
polymorphisms at the priming sites or even the absence of the ALS3 locus in these isolates.
Evidence of the spontaneous deletion in other genes of the same family, such as ALS5 and
ALS6 genes, was already observed by Hoyer & Hetch (2001) and Zhao et al. (2007).
In conclusion, we showed that Candida non-albicans species were more prolific
biofilm producers than C. albicans and that the source of isolates may influence the biofilm
production, in which more invasive isolates show greater biofilm productivity. The
polymorphism of the ALS3 central domain in C. albicans, detected by the different numbers of
tandem-repeat copies, appears not to be directly related to biofilm production.
Acknowledgements
We thank Drs Augusto Cezar Montelli, Terue Sadatsune and Alessandro Lia Mondelli,
for their help in providing the isolates, and Raquel Cordeiro Theodoro, Sandra Bosco, Severino
Assis Macoris and Virginia Richini-Pereira, for their lab assistance.
This work was supported by Fapesp (grant n º 2007/01946-4).
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Table 1
: Distribution of Candida species obtained from different sources in a Brazilian tertiary public
hospital (HC/UNESP), 1998-2005.
Species
identification
Blood Urine Vaginal
a
Peritoneal
b
Total
% (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
a
Vaginal secretion;
b
Peritoneal fluid
% of isolates
Score
Blood
Urine
Vaginal
Peritoneal
Figure 1
. Frequency distribution of Candida species in relation to the clinical sources and biofilm
production scores, categorized according to Tumbarello et al., 2007.
Table 2: Comparison of biofilm production by Candida species isolates from blood, urine, vaginal secretion and peritoneal fluid obtained in a Brazilian
tertiary public hospital (HC/UNESP), 1998-2005.
Candida species
No. of biofilm positive/no.total (%)
Total
Blood
Urine
Vulvov
Perit
C. albicans 66/153 (43.1) 13/23 (56.5) 12/29 (41.4) 34/93 (36.6) 7/8 (87.5)
All non-albicans species 132/174 (75.9)
a
68/79 (86.1)
a
42/53 (79.2)
a
9/22 (40.9)
a
13/17 (76.5)
C. parapsilosis
53/66 (80.3)
40/44 (90.9)
5/7 (71.4)
1/5 (20.0)
7/10 (70.0)
C. tropicalis 35/37 (94.6) 3/3 (100.0) 28/28 (100.0) 0/1 (0.0) 4/5 (80.0)
C. glabrata 13/29 (44.8) 3/5 (60.0) 10/19 (52.6) 1/4 (25.0) -
Other
Candida
species
31/42 (73.8)
24/27 (88.9)
-
7/12 (58.3)
2/2 (100.0)
Total 198/327 (60.6) 81/102 (79.4) 54/85 (63.5)
b
43/115 (37.4)
c
, d
20/25 (80.0)
a
P < 0.001, C. albicans versus non-albicans
b
P < 0.02, blood versus urine
c
P < 0.001, blood versus vaginal secretion
d
P < 0.001, peritoneal fluid versus vaginal secretion
1 2 3 4 5 6 7
2.0
1.5
1.0
Figure
2
.
Ethidium bromide
-
stained agarose gel highlighting amplification of PCR products from the
tandem repeat region of ALS3 gene of 6 C. albicans isolates with ALS3GenoF and ALS3genoR primers.1-
alleles with 10 and 13 motifs; 2- alleles with 10 and 11 motifs; 3- alleles with 10 motifs each; 4- alleles
with 11 motifs each; 5- alleles with 14 motifs each; 6- alleles with 11 motifs each; 7- Ladder 1kb
(Promega).
kb
Table 3: Distribution of allele per ALS3 gene tandem-repeat copies in C. albicans isolates from the bloodstream obtained in a Brazilian tertiary public
hospital (HC/UNESP), 1998-2005.
Biofilm production (n)
Percent (n) alleles in each tandem repeat copy group
Mean no of repeat copies/allele
a
7
10
11
12
13
14
Biofilm negative (16)
0
(0)
31.3 (5)
25.0 (4)
0 (0)
43.8 (7)
0 (0)
11.6 ± 1.4
Biofilm positive (22)
9.1 (2) 36.4 (8) 36.4 (8) 4.5 (1) 4.5 (1) 9.1 (2) 10.7 ± 1.7
a
Mean number of repeat copies per allele ± standard deviation. P value not significant.
CONSIDERAÇÕES FINAIS
Infecções noscomiais por leveduras constituem em sério problema de saúde pública,
com altos custos econômicos e sociais, e com forte evidência que tenderá a aumentar nos
próximos anos. Já está bem estabelecido que diferentes espécies, além de C. albicans, são os
agentes envolvidos neste processo. Tendo em vista que diferentes espécies apresentam
diferentes perfis epidemiológicos e de resposta aos tratamentos, é fundamental que se faça
estudos adequados de identificação e caracterização destes agentes, principalmente nos
hospitais terciários que atendem os pacientes particularmente mais susceptíveis.
A produção de biofilme é um fenômeno importante na patogenicidade de vários
microrganismos, e pode acarretar em várias implicações, tanto em ambientes hospitalares,
contaminando próteses, cateteres e equipamentos médicos, como industriais, aderidos a
equipamentos para processamento de água e alimentos. Atualmente sabe-se que a produção de
biofilme, tanto fúngico quanto bacteriano, compreende processos controlados geneticamente.
No entanto, enquanto os estudos genéticos e moleculares da produção de biofilme em C.
albicans avançaram significativamente, o mesmo ainda não ocorreu em outras espécies de
Candida, como por exemplo, C. parapsilosis e C. tropicalis, notáveis pela produção de
biofilme. Fundamental, portanto, aprofundar estes estudos de forma a proporcionar um melhor
conhecimento e entendimento das peculiaridades de cada espécie, bem como características
comuns entre as mesmas, que podem gerar métodos mais eficientes de controle.
Um melhor entendimento sobre o processo de formação e desenvolvimento do biofilme
associados a estudos mais aprofundados dos genes envolvidos podem proporcionar importantes
avanços terapêuticos e técnicas para minimizar ou até mesmo impedir infecções decorrentes da
formação de biofilme.
CONCLUSÕES
1. C. albicans continua sendo a espécies mais freqüente em infecções fúngicas no
Hospital das Clínicas da faculdade de Medicina de Botucatu-UNESP, embora, a
proporção de espécies não-albicans é maior que de C. albicans.
2. A grande maioria de C. albicans foi isoladas de infecções não invasivas, e C.
parapsilosis de infecções invasivas com diferença significativa em relação a C.
albicans.
3. De modo geral, as drogas antifúngicas utilizadas exerceram boa atividade sobre as
amostras estudadas.
4. A produção de biofilme foi mais positiva entre isolados de espécies não-albicans
em relação à C. albicans.
5. Isolados de infecções invasivas apresentaram maior positividade para a produção
de biofilme.
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