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MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
AVALIAÇÃO DA PREVALÊNCIA, FATORES DE VIRULÊNCIA E
SUSCEPTIBILIDADE ANTIFÚNGICA DE Candida spp. oriundas de fezes de
pombos (Columbia livia)
LUCIANA MAGALHÃES PINTO
NATAL/RN
2018
I
LUCIANA MAGALHÃES PINTO
AVALIAÇÃO DA PREVALÊNCIA, FATORES DE VIRULÊNCIA E
SUSCEPTIBILIDADE ANTIFÚNGICA DE Candida spp. oriundas de fezes de
pombos (Columbia livia)
Dissertação apresentada ao
Programa de Pós-Graduação em
Ciências da Saúde da
Universidade Federal do Rio
Grande do Norte como requisito
para a obtenção do título de
Mestre em Ciências da Saúde
Orientador: Prof. Dr. Guilherme
Maranhão Chaves
NATAL/RN
2018
I
Universidade Federal do Rio Grande do Norte - UFRN
Sistema de Bibliotecas - SISBI
Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS
Pinto, Luciana Magalhães.
Avaliação da prevalência, fatores de virulência e susceptibilidade
antifúngica de Candida spp. oriundas de fezes de pombos (Columbia livia) /
Luciana Magalhães Pinto. - 2018.
59f.: il.
Dissertação (Mestrado) - Universidade Federal do Rio Grande do Norte,
Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde.
Natal, RN, 2018.
Orientador: Prof. Dr. Guilherme Maranhão Chaves.
1. Candida spp. - Dissertação. 2. fatores de virulência - Dissertação. 3.
resistência antifúngica - Dissertação. 4. fezes de pombos - Dissertação. 5.
Columbia livia - Dissertação. I. Chaves, Guilherme Maranhão. II. Título.
RN/UF/BSCCS CDU 616.97
Elaborado por Adriana Alves da Silva Alves Dias - CRB-15/474
II
MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenadores do Programa de Pós-Graduação em Ciências da Saúde
Profa. Dra. Ana Katherine Gonçalves
Prof. Dr. Guilherme Maranhão Chaves
III
LUCIANA MAGALHÃES PINTO
AVALIAÇÃO DA PREVALÊNCIA, FATORES DE VIRULÊNCIA E
SUSCEPTIBILIDADE ANTIFÚNGICA DE Candida spp. oriundas de fezes de
pombos (Columbia livia)
Aprovada em ___/___/___
Banca examinadora:
Presidente da Banca:
Prof. Guilherme Maranhão Chaves (UFRN)
_______________________________________________________________
Examinador externo:
Profa. Raquel de Melo Barbosa (UNINASSAU)
Examinador externo ao Programa:
Profa. Eveline Pipolo Milan (UFRN)
_______________________________________________________________
Examinador interno:
Dr. Walicyranison Plinio da Silva Rocha (UFRN)
IV
AGRADECIMENTOS
Primeiramente agradeço a Deus, por ser essencial em minha vida, autor de meu
destino, meu guia, e sempre presente nas horas de angústias, iluminando meu caminho.
Agradeço aos meus pais (Sônia Magalhães e Wagner Pinto) por sempre me apoiarem
em minhas decisões, sonhando juntamente comigo, me dando forças para nunca desistir
dos meus sonhos e sempre persistir neles, me proporcionando tudo de melhor que eles
podiam me oferecer, e a toda minha família.
Agradeço aos Meus amigos pela paciência, devido a todos meus momentos de
estresses, pela minha falta de tempo em determinadas ocasiões, mas que sempre estiveram
comigo nos momentos não só de alegria, mas nos momentos mais difíceis. Não se
esquecendo dos meus agradecimentos aos Amigos que fiz durante toda minha graduação,
nossos momentos de estudos em grupo, ajudando sempre uns aos outros para que
pudéssemos vencer juntos cada desafio.
Ao Prof. Dr. Guilherme Maranhão Chaves, que me acolheu, no momento em
que fui mostrar meu interesse para fazer parte de sua equipe, e que ao longo desses dois
anos e meio, me incentivou, e me ajudou em todos os momentos que precisei. Ensinou-
me a ser uma pessoa mais responsável, a cumprir com todos os meus deveres, e me ajudou
a construir um crescimento pessoal.
A todos os meus companheiros de pesquisa que fizeram e/ou fazem parte do
LMMM: Luanda, Diana, Ana Patricia, Alanny, Débora, Karen, Vitor, Plínio e
Alysson, que se tornaram uma família, com nossa convivência diária, sempre buscando
trabalhar em equipe, ajudando uns aos outros, proporcionando nosso ambiente
harmônico.
V
RESUMO
Embora seja amplamente conhecido que Cryptococcus spp. podem transmitir
criptococose através de aerossol formado quando fezes secas (principalmente
de pombos) são dispersadas e aerotransportadas, pouco se sabe sobre o papel
dessas aves em abrigar outras leveduras patogênicas em seu trato
gastrointestinal, penas e bicos, especificamente porque esses animais
frequentemente permanecem e reproduzem-se próximas ou mesmo acima de
unidades de condicionadores de ar. Avaliou-se a prevalência de leveduras
patogênicas isoladas de fezes de pombos coletadas na área externa de um
hospital universitário no Brasil. Nosso objetivo foi também investigar o potencial
patogênico e a susceptibilidade antifúngica de espécies de Candida de interesse
médico isoladas dessas amostras. Portanto, foi realizada a avaliação da
expressão de atributos de fatores de virulência in vitro, incluindo a capacidade
de aderir às células epiteliais bucais humanas e a formação de biofilme e
produzir enzimas líticas, como fosfolipases, proteinases e hemolisinas. Testes
de sensibilidade antifúngica frente a fluconazol, itraconazol, anfotericina e
micafungina também foram realizados. O gênero Candida foi o mais prevalente
em nosso estudo, com várias espécies clinicamente importantes sendo isoladas.
Vale ressaltar que essas cepas foram capazes de expressar vários fatores de
virulência, mostrando claramente seu potencial patogênico. Esse estudo foi
capaz de demonstrar que Candida spp. isoladas de excrementos de pombos
podem expressar fatores de virulência da mesma forma de isolados clínicos,
sugerindo um potencial patogênico para estas leveduras. O fato de essas cepas
terem sido coletadas da área externa de um hospital terciário apresenta potencial
interesse, porque elas podem ser uma fonte de infecção, especificamente para
hospedeiros imunocomprometidos.
Palavras-Chave:
Candida spp., fatores de virulência, resistência antifúngica, fezes de pombos,
Columbia livia
VI
LISTA DE TABELAS
Tabela 1 - Prevalência de leveduras obtidas de amostras de excrementos de
pombos de áreas públicas da cidade de Natal, estado do Rio Grande do Norte,
Nordeste do Brasil.
Tabela 2 - Fatores de virulência de Candida spp. isolados de fezes de pombos
da cidade de Natal, estado do Rio Grande do Norte, Nordeste do Brasil.
Tabela 3 - Comparações médias de fatores de virulência de Candida spp.
isolados de excrementos de pombos da cidade de Natal, estado do Rio Grande
do Norte, Nordeste do Brasil.
Tabela 4 - Resultados do teste de sensibilidade de Candida spp. isolados de
fezes de pombos da cidade de Natal, estado do Rio Grande do Norte, Nordeste
do Brasil.
VII
SUMÁRIO
1. INTRODUÇÃO.............................................................................................. 8
2. JUSTIFICATIVA............................................................................................10
3. OBJETIVOS..................................................................................................11
4.MATERIAIS E MÉTODOS.............................................................................12
4. Coleta e processamento de amostras fecais de pombos ............................12
4.2 Identificação das leveduras isoladas..........................................................12 4.3 Amostras utilizadas no presente estudo.....................................................13 4.4 Padronização do Inoculo para Candida spp. Fatores de virulência avaliados
in vitro...............................................................................................................13
4.5 Adesão às Células Epiteliais Bucais Humanas de (HBEC) de Candida
spp....................................................................................................................14
4.6 Formação de biofilme de Candida spp.......................................................14
4.7 Atividade de Proteinase..............................................................................15
4.8 Atividade de Hemolisina.............................................................................16
4.9 Atividade de fosfolipase..............................................................................16
4.10 Perfil de suscetibilidade antifúngica de Candida spp................................17
5. ARTIGOS PRODUZIDOS.............................................................................19
5.1 Candida species isolated from pigeon (Columbia livia) droppings may express
virulence factors and resistance to azoles
6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES..............................................43
7. REFERÊNCIAS.............................................................................................44
8. APÊNDICE ....................................................................................................46
8
1. INTRODUÇÃO
Aves selvagens, incluindo pombos (Columbia livia), têm sido
consideradas uma fonte importante de leveduras patogênicas (1). No Brasil, a
população de pombos domésticos aumentou significativamente, tornando-se um
problema ambiental e influenciando a saúde pública (2). Além da colonização
microbiana em seus bicos, pés e penas, os pombos podem disseminar leveduras
patogênicas para o meio ambiente e os seres humanos, principalmente a partir
do aerossol presente nas fezes secas (1).
Diversos dados epidemiológicos foram publicados sobre o isolamento de
leveduras de fezes de pombos em todo o mundo (1, 2, 3). Embora a maioria dos
estudos nesta área se concentre na avaliação da prevalência de leveduras
pertencentes ao gênero Cryptococcus (4), leveduras de outros gêneros, como
Candida, Rhodotorula e Trichosporon podem ser isoladas de excrementos de
pombos e podem ter o potencial para causar infecções fúngicas, incluindo
doenças invasivas que ameaçam a vida (2,5).
Em um estudo realizado por Jang et al. (6) em Seul, Coréia, 306 amostras
de fezes foram coletadas de parques e quadras. Cento e vinte e seis amostras
levaram ao isolamento de leveduras. Candida glabrata foi a espécie mais
prevalente (34,1%), seguida de C. famata (12,7%). C. krusei e Trichosporon
asahii (1,16%) foram isolados em menor número. Mais recentemente, outro
estudo realizado na Ilha de Gran Canaria (Espanha) focou no isolamento de
leveduras não-Cryptococcus de 83 amostras de fezes de pombos e encontrou
C. guilliermondii e C. albicans como as espécies mais frequentemente isoladas
(49,4 e 15,7% das amostras, respectivamente). Outras importantes leveduras de
interesse médico, como Trichosporon spp. e Rhodotorula mucilaginosa também
foram recuperadas (1).
Candida spp. podem fazer parte da microbiota humana normal, sendo
que o desenvolvimento de candidíase invasiva é principalmente de origem
endógena. No entanto, infecções exógenas estão ganhando notoriedade (7).
Neste contexto, aves domésticas e silvestres podem servir como reservatórios
9
para leveduras com alto potencial patogênico, dispersas pelo ar, servindo como
fonte de colonização e causando infecções graves com alta taxas de mortalidade
(1).
Os principais fatores de virulência de Candida spp. incluem a capacidade
de aderir às células epiteliais bucais humanas (CEBH) e células endoteliais, a
transição levedura-hifas (morfogênese, um fenótipo típico de C. albicans), a
produção de enzimas hidrolíticas extracelulares (proteinases, hemolisinas e
fosfolipases), formação de biofilme e a capacidade de evitar o ataque de células
do sistema imunológico (8).
Estudos sobre a investigação da expressão in vitro de fatores de virulência
de Candida spp. isoladas de excrementos de pombos são praticamente
inexistentes. No entanto, Jang et al., (6) realizando uma investigação
semelhante, descobriram que 6 isolados de C. glabrata e um único isolado de C.
krusei, oriundos de amostras fecais de pombos em Seul, Coréia, demostraram
atividade proteinase.
Devido aos fatores previamente mencionados, é importante conhecer o
potencial patogênico das leveduras, podendo estas servirem de fonte de
colonização e causar mais candidíase invasiva, avaliando especificamente a
expressão dos fatores de virulência in vitro e sua possível relação com o
processo de patogênese das infecções causadas por Candida spp. Além disso,
a determinação do perfil de suscetibilidade antifúngica desses isolados obtidos
do ambiente próximo a um hospital universitário terciário é de fundamental
importância para a implementação de terapêuticas mais efetivas e para o
estabelecimento de abordagens de controle de infecção nosocomial. Portanto,
os principais objetivos deste estudo foram avaliar a patogenicidade e a
susceptibilidade antifúngica de espécies de Candida isoladas de fezes de
pombos de uma área externa de um hospital terciário da cidade de Natal, Estado
do Rio Grande do Norte, Brasil. Para o melhor conhecimento, este é o primeiro
estudo no mundial a demonstrar que as leveduras não Cryptococcus isoladas de
excrementos de pombos podem expressar fatores de virulência in vitro e exibir
resistência a drogas antifúngicos azólicos sem possíveis exposições prévias a
estes compostos químicos.
10
2. JUSTIFICATIVA
Além do conhecimento sobre a distribuição dos fungos de interesse
médico no ambiente, torna-se importante verificar a capacidade dos mesmos em
causar doença (patogenicidade). A transição de um micro-organismo comensal
inofensivo a patógeno é atribuível a um extenso repertório de fatores de
virulência seletivamente expressos sob condições predisponentes. Este fator
ressalta a natureza oportunista das infecções por leveduras, onde o micro-
organismo que faz parte da microbiota normal pode tornar-se patogênico (9).
O uso indiscriminado de antifúngicos, especialmente em pacientes
imunocrompometidos, mas também na prática veterinária e na agricultura,
contribui para o surgimento de resistência a essas drogas (10). Outra justificativa
para o estudo das infecções provocadas por estas leveduras é que, embora
existam muitos compostos antifúngicos disponíveis no mercado, tais drogas têm
limitações em sua utilização, por apresentarem toxicidade ao hospedeiro, e
também pela possibilidade de ocorrer a manifestação de resistência, por
diferentes espécies de Candida, como C. krusei, intrinsicamente resistente ao
fluconazol e C. lusitaniae à anfotericina B (11).
A análise da similaridade genética entre diferentes cepas de uma mesma
espécie de micro-organismo patogênico é ferramenta fundamental para permitir
a investigação da distribuição geográfica destas espécies, caracterizar suas
fontes e mecanismos de transmissão, monitorar a emergência de cepas
resistentes a drogas e auxiliar na definição de surtos (12).
Há poucos dados na literatura concernentes à patogenicidade,
epidemiologia molecular e susceptibilidade aos antifúngicos de isolados clínicos
e ambientais de Candida spp. obtidos no nordeste brasileiro, inclusive no estado
do Rio Grande do Norte, onde, pelo nosso conhecimento, não há estudos dessa
natureza. Mais estudos também se fazem necessários para melhor elucidar a
relação entre os genótipos de Candida spp. analisando a variabilidade biológica
intraespecífica para detectar polimorfismos genéticos em espécies de leveduras,
os quais podem estar correlacionados com a virulência e susceptibilidade aos
antifúngicos, podendo constituir fontes de infecção.
11
3. OBJETIVOS
3.1 Objetivos gerais:
Esse estudo tem objetivo geral avaliar a prevalência de leveduras isoladas
de fezes de pombos em um hospital terciário da cidade do Natal-RN, Brasil, bem
como realizar extensa caracterização fenotípica de Candida spp. quanto a
expressão in vitro de fatores de virulência e susceptibilidade a antifúngicos
sintéticos.
3.2 Objetivos específicos:
a) Isolar e identificar através de taxonomia clássica e MALDI-TOF/MS
cepas de Candida spp. obtidas a partir de amostras de fezes de
pombos dos arredores de um Hospital Universitário, da cidade de
Natal-RN, Brasil;
b) Caracterizar fenotipicamente as referidas cepas quanto aos fatores de
virulência, incluindo produção de fosfolipase e proteinase, formação
de biofilme, capacidade de adesão às células epiteliais e atividade
hemolítica;
c) Avaliar o perfil de susceptibilidade aos antifúngicos anfotericina B,
fluconazol, itraconazol e micafungina das referidas cepas.
12
4. MATERIAIS E MÉTODOS
4.1 Coleta e processamento de amostras fecais de pombos
O estudo incluiu 19 amostras de excrementos de pombos coletadas fora
do prédio de um hospital universitário terciário, incluindo as janelas das
enfermarias, localizadas na cidade de Natal, estado do Rio Grande do Norte,
Brasil, de abril de 2012 a março de 2014.
Aproximadamente 1 g de fezes de pombas secas e/ou frescas foram
coletadas e diluídas em solução salina (NaCl 0,9%) em uma proporção de 1: 9,
agitadas em um agitador mecânico por 10 minutos e deixadas em repouso por
1h. Posteriormente, 100 μL do sobrenadante foram removidos e inoculados em
duas placas CHROMagarCandida® (CHROMagar ™ Candida, Difco, EUA) com
o auxílio de uma alça de Drigalski. Cada placa foi incubada a 30 °C e 37 °C
durante 96 h. Colônias com características fenotípicas distintas (aparência, cor,
tamanho) foram selecionadas para posterior identificação, proporcionalmente à
quantidade total apresentada em cada amostra.
4.2 Identificação de leveduras isoladas
Após o crescimento das culturas, as leveduras foram novamente plaqueadas
em CHROMagar Candida (CHROMagar Microbiology, Paris, França) para
verificar a pureza e o rastreio de colônias de cores diferentes (13). A identificação
das espécies baseou-se nas características das células observadas
microscopicamente após o cultivo em ágar fubá adicionado de Tween 80, bem
como pela metodologia clássica (Yarrow, 1998) e pelo Sistema ID32C
(bioMerieux Marcy l'Etoile, França), sempre que necessário. Após a identifição,
13
todas as cepas de levedura foram cultivadas em caldo YPD (Peptone Dextrose
de levedura, extrato de levedura 10 g/L, 20 g/L de dextrose, peptona 20g/L)
“overnight” a 30 e depois transferidas para criotubos contendo 20% de glicerol e
armazenados a -80 ° C. Os isolados pertencem ao banco de microrganismos do
Laboratório de Micologia Molecular e Molecular, do Departamento de Análises
Clínicas e Toxicológicas, da Universidade Federal do Rio Grande do Norte.
4.3 Amostras utilizadas no presente estudo
Foram selecionados 60 isolados de Candida spp. da nossa coleção de
culturas que engloba todas as leveduras obtidas de fezes de pombos para
realizar a avaliação da expressão de fatores de virulência in vitro a saber:
complexo C. parapsilosis (n = 24), C. tropicalis (n = 19), C. krusei (n = 7), C.
glabrata (n = 4) e C. rugosa (n = 1). As cepas de referência C. parapsilosis
ATCC22019, C. tropicalis ATCC13803, C. krusei ATCC6258, C. glabrata ATCC
2001 e C. rugosa ATCC10571 foram utilizadas como controles para todos os
testes de identificação e determinação dos atributos de virulência in vitro e testes
de susceptibilidade antifúngica.
4.4 Padronização do inóculo de Candida spp. para a avaliação dos fatores de
virulência avaliados in vitro
Para a caracterização fenotípica dos diferentes isolados, as cepas foram
inicialmente cultivadas em caldo NGY (Difco Neopeptone 1 g/L, Dextrose 4 g / L;
Difco extrato de levedura 1 g / L). As células foram inoculadas por "wet looping"
neste meio (com o anel da alça carregado com um filme de suspensão de
leveduras rapidamente imerso no meio e removido) e incubadas por 18-24h em
agitador a 30 ° C, 200 rpm, sendo um inóculo de aproximadamente 2x108 células/
14
mL produzido (14). A densidade óptica celular foi medida a um comprimento de
onda de 600 nm variando de 0,8 e 1,2, através de espectrofotômetro (Biochrom
Libra S32). Posteriormente, as células de leveduras foram diluídas para obter o
inóculo específico necessário para cada atributo de virulência avaliado in vitro.
4.5 Adesão de Candida spp. às células epiteliais bucais humanas (CEBH)
As células de Candida spp. foram crescidas “overnight” até a fase
estacionária em NGY (0,1% de Neopeptona [Difco], 0,4% de glucose e 0,1% de
Extrato de Levedura [Difco]) a 30 ° C e foram misturadas com células epiteliais
bucais humanas (CEBH) de voluntários saudáveis a uma proporção de 10
células de levedura por cada CEBH. As suspensões celulares foram incubadas
a 37 °C durante 1 h com agitação mecânica (200 rpm). Posteriormente, as
células foram agitadas em vórtex, fixadas em formalina e transferidas para uma
lâmina de microscópio. O número de células de Candida spp. aderidas a 150
CEBH foi determinado na lâmina através de microscopia óptica (400x de
magnificação). Os testes foram realizados em triplicata (8).
4.6 Formação de biofilme de Candida spp.
Os ensaios de formação de biofilme foram realizados segundo Melo et al.
(2011). Inicialmente, alíquotas de 100 μL de uma suspensão celular padronizada
(107 células / mL) foram transferidas para placas de microtitulação de 96 poços
de fundo chato e incubadas por 1,5 h a 37 ° C em um agitador a 75 rpm. Como
controles, foram manipulados oito poços de cada placa de microtítulo de maneira
idêntica, com a excepção de não terem sido adicionadas suspensões de células
Candida. Após a fase de adesão, as suspensões de células foram aspiradas e
cada poço lavado duas vezes com 150 μL de PBS para remover células
15
fracamente aderidas. Um total de 100 μL de meio YNB (“Base de Nitrogênio de
Levedura”, DifcoTM) com 50 mM de glicose (D-glicose monohidratada PA,
Cinética) foi adicionado a cada um dos poços lavados e incubado a 37 ° C em
um agitador a 75ºC. rpm. Os biofilmes foram formados por 72 horas e
quantificados pelo ensaio de cristal violeta. Resumidamente, os poços revestidos
com biofilme de placas de microtitulação foram lavados duas vezes com 150 µl
de PBS e depois secos ao ar livre durante 45 min. Subsequentemente, cada um
dos poços lavados foi corado com 110 μl de solução aquosa de cristal violeta a
0,4% durante 45 min. Em seguida, cada poço foi lavado quatro vezes com 350
μL de água destilada estéril e posteriormente adicionados 200 μL de etanol a
95%. Após 45 min, 100 μL da solução de descoloração foram transferidos para
um novo poço e a quantidade de cristal violeta da solução referida medida com
um leitor de placas de microtitulação (SpectraMAX 340 Tunable Microplate
Reader; Molecular Devices Ltda.) a 570 nm. Os valores de absorbância para os
controles foram subtraídos dos valores dos poços teste para minimizar o
“background”. A interpretação da produção de biofilme seguiu os critérios
descritos por Stepanovic et al. (15).
4.7 Produção de Proteinase
A atividade de proteinase foi determinada pelo método descrito por Zuza-
Alves et al., (8). Alíquotas contendo 50 µL de culturas crescidas em NGY foram
adicionadas a 5 ml de meio YCB + ASB (11,7 g/L de “yeast carbon base” [Difco];
10 g /L de glucose; 5 g / L de albumina de soro bovino (ASB), fracção V [Sigma-
Aldrich]) sob agitação mecânica a 30 ° C durante 72 h, 200 rpm. A atividade
proteolítica foi determinada medindo-se o aumento em produtos solúveis em
ácido tricloroacético com absorbância a 280 nm em triplicata, após 1 h de
16
incubação do sobrenadante da cultura em substrato de ASB a 37 ° C. A atividade
enzimática foi expressa como a seguinte fórmula: DO280nm/DO600nm da
cultura. Leituras de DO iguais ou inferiores a 0,02 foram consideradas abaixo do
limite de detecção da técnica, sendo representadas como negativas.
4.8 Produção de Hemolisina
Para avaliar a produção de hemolisina, seguimos a metodologia proposta por
Luo et al. (16) com algumas adaptações. As células de levedura foram
inicialmente cultivadas em ASD a 35 ° C durante 18 h. As amostras foram
cultivadas em caldo NGY. Dez microlitros de cultura celular foram semeados em
triplicata na superfície de ASD contendo 7% de sangue de carneiro fresco (Ebe-
Farma) e 3% de glicose, contidos em placas de Petri de 155 mm de diâmetro.
As placas foram incubadas durante 48 h a 37 °C numa atmosfera com 5% de
CO2. Após o período de incubação, a presença de um halo claro ao redor do
inóculo indicou hemólise positiva. O diâmetro das colônias e zonas de hemólise
foram medidos para se obter o índice de hemólise (IH) para cada cepa. O IH foi
determinado dividindo-se o diâmetro da colônia pela soma da zona de hemólise
mais o diâmetro da colônia, o que permitiu classificar os isolados em produtores
fortes, moderados e fracos, segundo Linares et al. (17). Como controle positivo,
utilizou-se uma cepa beta hemolítica de Streptococcus pyogenes (Grupo A). A
cepa de referência de Candida parapsilosis ATCC22019 foi usada como controle
negativo para a produção de hemólise (16).
4.9 Produção de fosfolipase
Para detecção da atividade da fosfolipase, o método de Price et al. (18) foi
utilizado. As culturas em NGY crescidas “overnight” foram diluídas e
17
padronizadas para uma concentração de 2 x 105 células / mL; e a suspensão de
células inoculada em triplicata na superfície de Ágar Fosfolipase (10 g de
peptona, 40 g de dextrose, 16 g de ágar, 80 mL de Emulsão de Gema de Ovo
[Fluka]adicionada a 1000 mL de água destilada). As placas foram incubadas a
30°C por 72 h. Após o período de incubação, os diâmetros das colônias e o halo
formado ao redor deles foram medidos. O Pz (zona de fosfolipase) foi
determinado dividindo-se o diâmetro da colônia pela zona de precipitação mais
o diâmetro da colônia. Os isolados foram classificados da seguinte maneira, de
acordo com a distribuição dos tercis: Pz = 1 como atividade negativa da
fosfolipase; 0,82 ≤ Pz ≤ 0,88 como de produção fraca; 0,75 ≤ Pz ≤ 0,81 como
moderados; 0,67 ≤ Pz ≤ 0,74 como produtores fortes de fosfolipase.
4.10 Perfil de susceptibilidade antifúngica de Candida spp.
Soluções de fluconazol (FLU), itraconazol (ITC), micafungina (MCF) e
anfotericina B (AMB) foram preparadas de acordo com as diretrizes do
documento M27-A3 (18) sendo diluídas em RPMI 1640 (Roswell Park Memorial
Institute) (Angus buffers e Bioquímico, Niagara Falls, NY, EUA) tamponados com
ácido 3- (N-morfolino) propanossulfônico (MOPS) a pH 7,0. As drogas
antifúngicas testadas foram diluídas em série em 10 diferentes concentrações, a
saber: FLU (Pfizer Incorporated, Nova York, NY, EUA) para 0,125-64 μg/mL; ITC
(Pfizer Incorporated, Nova Iorque, NY, EUA), MCF (Merck, Rahway, NJ, EUA);
e AMB (Sigma Chemical Corporation, St. Louis, MO, EUA) para 0,015-8 μg / mL.
O inóculo de todas as cepas testadas foi obtido a partir de 24 h de cultivo em
ASD 35°C e uma suspensão inicial preparada com 90% de transmitância a 530
nm. Em seguida, foram realizadas duas diluições seriadas, a primeira em
solução salina (1: 100) e a segunda em RPMI (1:20), para obtenção da
18
concentração final de 103 células / mL. A suscetibilidade a agentes antifúngicos
foi avaliada por microdiluição em caldo, conforme recomendado no documento
CLSI M27-A3 (18). Alíquotas de 100 μL da solução final do inóculo foram
distribuídas em placas de microtitulação de 96 poços contendo 100 μL de várias
concentrações dos fármacos testados. Por fim, as placas foram incubadas a 37
° C e a leitura do teste foi feita após 24 h de incubação para equinocandinas e
fluconazol, e após 48 horas para os outros azóis e AMB (19,20). A CIM foi
definida para os azólicos e equinocandinas como a menor concentração do
fármaco que demostrou uma redução de cerca de 50% na turbidez em
comparação com o poço de controle positivo. Para a AMB, a CIM foi definida
como a menor concentração capaz de inibir qualquer crescimento visualmente
perceptível (20).
Análise Estatística
Os dados foram analisados por meio do programa estatístico “Graph Pad,
Prism” versão 6.0 e “Stata” versão 11.0. Os resultados foram apresentados como
média ± desvio padrão, e as diferenças foram analisadas pelo teste T de Student.
Para todas as análises, valores de P <0,05 foram considerados significantes e o
intervalo de confiança de 95% foi selecionado. Além disso, todos os valores
obtidos para alguns testes de atributos de virulência in vitro foram divididos em
categorias de tercil como produtores fracos, moderados ou fortes.
19
5. ARTIGOS PRODUZIDOS
5.1 Candida species isolated from pigeon (Columbia livia) droppings may
express virulence factors and resistance to azoles
Periódico: Veterinary Microbiology
ISSN: 0378-1135
Qualis:
Fator de Impacto: 2.524
Status:Submetido
20
Candida species isolated from pigeon (Columbia livia) droppings may
express virulence factors and resistance to azoles
Luciana Magalhães Pinto1, Francisco de Assis Bezerra Neto1, Mariana
Araújo Paulo de Medeiros1, Diana Luzia Zuza Alves1, Guilherme Maranhão
Chaves1*.
1 Laboratory of Medical and Molecular Mycology, Department of Clinical
and Toxicological Analyses, Federal University of Rio Grande do Norte, Natal,
Rio Grande do Norte, Brazil.
* Author responsible for correspondence:
Name: Guilherme Maranhão Chaves
Address: Universidade Federal do Rio Grande do Norte, Centro de Ciências
da Saúde. Departamento de Análises Clínicas e Toxicológicas. Laboratório de
Micologia Médica e Molecular. Rua. Gal. Gustavo Cordeiro de Faria S/N.
Petrópolis. Natal, RN – Brasil. CEP: 59012-570.
Phone number: 00 55 (84) 3342-9801
E-mail address: [email protected]
21
Abstract
Even though it is widely known that Cryptococcus spp. may transmit
cryptococcosis trough aerosol formed when dried birds (mainly pigeons)
droppings are dispersed and become airborne, little is known about the role of
these birds in harboring pathogenic yeasts in their gastrointestinal tract, feathers
and beaks, specifically because these animals often stay and reproduce close or
even above air conditioner units. Here we evaluated the prevalence of pathogenic
yeasts isolated from pigeon droppings collected in the outside area of a University
Hospital in Brazil. We also aimed to investigate the pathogenic potential and
antifungal susceptibility of Candida species of medical interest isolated from
these samples. Therefore, we performed the evaluation of virulence factors
attributes expression in vitro, including the ability to adhere to human buccal
epithelial cells and biofilm formation and to produce lytic enzymes, such as
phospholipases, proteinases and hemolysins. Antifungal susceptibility testing
against fluconazole, itraconazole, amphotericin and micafungin were also
performed. The Candida genus was the most prevalent in our study, with several
medically important species being isolated. Of note, these strains were able to
express several virulence factors in vitro, clearly showing their pathogenic
potential. Our study was able to demonstrate that Candida spp. isolated from
pigeon droppings may express virulence factors in the same manner of clinical
isolates, suggesting a pathogenic potential for these yeasts. The fact these
strains were collected from the outside area of a tertiary hospital may be of
interest, because they may be a source of infection, specifically to
immunocompromised hosts.
Key-words: Candida spp, virulence factors, antifungal resistance, pigeon
droppings, Columbia livia
22
Introduction
Wild birds, including pigeons (Columbia livia), have long been considered a
major source of pathogenic yeasts (Rosario et al., 2017). In Brazil, the domestic
pigeon population has increased significantly, becoming an environmental
problem and influencing public health (Costa et al., 2010). In addition to microbial
colonization in their beaks, feet and feathers, pigeons can spread pathogenic
yeasts to the environment and humans mainly from aerosol present in dried
droppings Rosario et al., 2017).
Several epidemiological data have been published regarding the isolation
of yeasts from pigeon droppings around the world (Rosario et al.,2017; Wu et al.,
2012). Although most studies in this area focus on the evaluation of the
prevalence of yeasts belonging to the genus Cryptococcus (Teodoro et al., 2012),
yeasts of other genera, such as Candida, Rhodotorula and Trichosporon may be
isolated from pigeon droppings and could have the potential to cause fungal
infections including invasive life-threatening diseases (Cafarchia et al., 2006a;
Costa et al.,2010).
In a study performed by Jang et al. (2011) in Seoul, Korea, 306 faeces
samples were collected from public squares and courts. One hundred and twenty
six samples leaded to yeasts isolation. Candida glabrata was the most prevalent
species (34.1%), followed by C. famata (12.7%). C. krusei and Trichosporon
asahii (1.16%) were isolated in lower numbers. More recently, another study
performed in Gran Canaria Island (Spain) focused on the isolation of non-
Cryptococcus yeasts from 83 pigeon droppings samples and found C.
guilliermondii and C. albicans as the most frequently isolated species (49.4 and
15.7% of the samples, respectively). Other important yeasts of medical interest,
such as Trichosporon spp. and Rhodotorula mucilaginosa were also recovered
(Rosario et al., 2017).
Because Candida spp. may be part of the normal human microbiota, the
development of invasive candidiasis is mostly of endogenous origin. However,
exogenous infections are gaining notoriety (Nucci et al., 2010). In this context,
23
domestic and wild birds may serve as reservoirs for yeasts with high pathogenic
potential, dispersed through the air and may further colonize and cause severe
infections with high mortality rates (Rosario et al., 2017).
The main Candida spp. virulence factors include the ability to adhere to
human buccal epithelial cells (HBEC) and endothelial cells, the yeast-to-hyphae
transition (morphogenesis, a typical phenotype of C. albicans), the production of
extracellular hydrolytic enzymes (proteinases, hemolysins and phospholipases),
biofilm formation, and the ability to evade the attack of immune system cells
(Zuza-Alves et al., 2017).
Studies concerning the investigation of the in vitro expression of virulence
factors by Candida spp. isolated from pigeon droppings are practically
nonexistent. However, Jang et al., (2011) performing a similar investigation found
that 6 isolates of C. glabrata and a single isolate of C. krusei, isolated from
pigeon’s fecal samples in Seoul, Korea, showed proteinase activity.
Because of the previously mentioned factors, it is important to know the
pathogenic potential of yeasts the may be a source of colonization and further
cause invasive candidiasis, specifically evaluating the expression of virulence
factors in vitro and its possible relationship with the pathogenesis process of
infections caused by Candida spp. Besides, the determination of drugs
susceptibility profiling of these isolates obtained from the environment nearby a
tertiary University hospital are of fundamental importance in order to implement
more effective therapeutics and to establish nosocomial infection control
approaches. Therefore, the main objectives of this study were to evaluate the
pathogenicity and antifungal susceptibility of Candida species isolated from
pigeon droppings from an external area of a tertiary hospital in the city of Natal,
Rio Grande do Norte State, Brazil. To the best of knowledge, this is the first study
in the world to demonstrate that non-Cryptococcus yeasts isolated from pigeon
droppings may express virulence factors in vitro and exhibit resistance to azole
antifungal drugs without previous known expositions to these chemical
compounds.
24
MATERIALS AND METHODS
Collection and processing of fecal samples from pigeons
The study included 19 samples of pigeon droppings collected outside the
building of a tertiary university hospital, including the windows of the wards,
located in Natal City, Rio Grande do Norte State, Brazil, from April 2012 to March
2014.
Approximately 1g of dried and/or fresh pigeon stools were collected and
diluted in saline solution (NaCl 0.9%) in a ratio of 1: 9, shaken on a mechanical
stirrer for 10 minutes and allowed to stand for 1h. Subsequently, 100 μL of the
supernatant was removed and inoculated into two CHROMagarCandida® plates
(CHROMagar ™ Candida, Difco, USA) with the aid of a Drigalski loop. Each plate
was incubated at 30 °C and 37°C for 96h. Colonies with distinct phenotypic
characteristics (appearance, color, size) were selected for subsequent
identification, proportionally to the total amount presented in each sample.
Identification of isolated yeasts
After cultures growth, yeasts were again plated on CHROMagar Candida
(CHROMagar Microbiology, Paris, France) to check for purity and screening for
different color colonies (Baumgartner et al., 1996). Species identification was
based on the characteristics of the cells observed microscopically after cultivation
on corn meal agar added Tween 80, as well as classical methodology (Yarrow,
1998) and ID32C System (bioMerieux Marcy l’Etoile, France), whenever it was
necessary. After identification, all yeast strains were cultured in YPD broth (Yeast
Peptone Dextrose, yeast extract 10 g / L, dextrose 20g / L, peptone 20g / L)
overnight at 30 ° C, and then were transferred to cryotubes containing 20%
glycerol and stored at -80 ° C. The isolates belong to the fungi culture collection
of the Medical and Molecular Mycology Laboratory, Department of Clinical and
Toxicological Analyses, Federal University of Rio Grande do Norte.
Strains used in the present study
25
We screened 60 isolates of Candida spp. from our culture collection to
perform the evaluation of the expression of virulence factors in vitro as follows:
C. parapsilosis complex (n=24), C. tropicalis (n=19), C. krusei (n=7), C. glabrata
(n=4) and C. rugosa (n=1). Of note, C. parapsilosis ATCC22019, C. tropicalis
ATCC13803, C. krusei ATCC6258, C. glabrata ATCC 2001 and C. rugosa
ATCC10571 and were used as controls for all the identification tests and the
determination of virulence attributes in vitro and antifungal susceptibility testing.
Inoculum Standardization for Candida spp. Virulence Factors
Evaluated In vitro
. For the phenotypic characterization of the different isolates, the strains
were initially grown in NGY broth medium (Difco Neopeptone 1 g/L, Dextrose 4
g/L; Difco yeast extract 1 g/L). When cells are inoculated by “wet looping” in this
medium (with a ring loop loaded with a yeast suspension film rapidly immersed
in the medium and removed) and incubated for 18-24h in shaker at 30°C, 200
rpm, an inoculum of approximately 2x108 cells/mL is produced (Chaves et al.,
2013). Cultures were spectrophotometrically measured at a wavelength of 600
nm ranging from 0.8 and 1.2 (Biochrom Libra S32). Subsequently, Candida spp.
cells were diluted to obtain the specific inoculum needed for each attribute of
virulence evaluated in vitro.
Candida spp. Adherence to Human Buccal Epithelial Cells (HBEC)
Candida spp. cells were grown overnight to stationary phase in NGY (0.1%
Neopeptone [Difco], 0.4% glucose and 0.1% Yeast Extract [Difco]) at 30°C and
were mixed with human buccal epithelial cells (HBEC) from healthy volunteers at
a ratio of 10 yeast cells per HBEC. The mixtures were incubated at 37°C for 1 h
with shaking; then cells were vortexed, formalin-fixed and transferred to a
microscope slide. The number of Candida spp. cells adhered to 150 HBEC was
determined with the operator blinded to the nature of the material on the slide.
Tests were done in triplicate (Zuza-Alves et al., 2017).
26
Candida spp. Biofilm Formation
Biofilm formation assays were performed according by Melo et al. (2011).
At first, 100 μL aliquots of a standardized cell suspension (107 cells/mL) were
transferred to flat bottom 96 well microtiter plates and incubated for 1.5 h at 37°C
in a shaker at 75 rpm. As controls, eight wells of each microtiter plate were
handled in an identical fashion, except that no Candida cell suspensions were
added. Following the adhesion phase, cell suspensions were aspirated and each
well was washed twice with 150 μL of PBS to remove loosely adherent cells. A
total of 100 μL of YNB medium (“Yeast Nitrogen Base”, DifcoTM) with 50 mM of
glucose (D-glucose monohidratada P.A., Cinética) was added to each of the
washed wells and incubated at 37°C in a shaker at 75 rpm. Biofilms were allowed
to develop for 66 h and quantified by the crystal violet assay. Briefly, the biofilm-
coated wells of microtiter plates were washed twice with 150 μL of PBS and then
air dried for 45 min. Subsequently, each of the washed wells was stained with
110 μL of 0.4% aqueous crystal violet solution for 45 min. Afterward, each well
was washed four times with 350 μL of sterile distilled water and immediately
distained with 200 μL of 95% ethanol. After 45 min, 100 μL of destaining solution
was transferred to a new well and the amount of the crystal violet stain in the
referred solution was measured with a microtiter plate reader (SpectraMAX 340
Tunable Microplate Reader; Molecular Devices Ltda.) at 570 nm. The absorbance
values for the controls were subtracted from the values for the test wells to
minimize background interference. Interpretation of biofilm production was
according to the criteria described by Stepanovic et al. (2007).
Candida spp. Proteinase Production
Proteinase activity was determined by a method Zuza-alves et al., 2017.
Fifty-microliter samples from NGY cultures were grown in 5 mL YCB + BSA
medium (11.7 g/L Yeast Carbon Base [Difco]; 10 g/L glucose; 5 g/L bovine serum
albumin, fraction V [Sigma–Aldrich]) rotated in a rotator shaker at 30°C for 72 h,
200 rpm. Proteolytic activity was determined by measuring the increase in
trichloroacetic acid soluble products absorbing at 280 nm in triplicate after 1 h
incubation of the culture supernatant with BSA substrate at 37°C. Specific activity
27
was expressed as OD280nm/OD600 nm of the culture. OD readings equal to or
below 0.02 were considered below the limit of detection of the technique and
were represented as negative.
Candida spp. Hemolysin Production
In order to evaluate hemolysin production, we followed the methodology
proposed by Luo et al. (2001) with some adaptations. Candida spp. cells were
initially cultured on SDA at 35°C for 18 h. Strains were next grown overnight in
NGY broth. Ten microliters of cell culture were seeded in triplicate on the surface
of SDA containing 7% fresh sheep blood (Ebe-Farma) and 3% glucose, contained
in Petri dishes of 155 mm of diameter. The plates were incubated for 48 h at 37°C
in an atmosphere with 5% CO2. After the incubation period, the presence of a
clear halo around the inoculum indicated positive hemolysis. The diameter of
colonies and zones of hemolysis were measured in order to obtain the hemolysis
index (HI) for each strain. HI was determined by dividing the colony diameter by
the precipitation zone plus colony diameter, which allowed classification of
isolates in strong, moderate and weak producers, according to Linares et al.
(2007). As a positive control we used a beta hemolytic strain of Streptococcus
pyogenes (Group A). The reference strain of Candida parapsilosis ATCC22019
was used as a negative control (Luo et al., 2001).
Candida spp. Phospholipase Production
For detection of the phospholipase activity, the method of Price et al. (1982)
was used. Overnight NGY cultures were diluted and standardized to a
concentration of 2×105 cells/mL; and the suspension of cells was inoculated in
triplicate on the surface of Phospholipase agar (10 g peptone, 40 g dextrose, 16
g agar, 80 mL Egg Yolk Emulsion [Fluka] was added to 1000 mL of distilled water
1000 mL). The plates were incubated at 30°C for 72 h. After the incubation period,
the diameters of the colonies and the halo formed around them were measured.
The Pz (phospholipase zone) was determined by dividing the colony diameter by
the precipitation zone plus colony diameter. The isolates were classified as
follows, according to tertiles distribution: Pz = 1 as negative phospholipase
28
activity; 0.82 ≤ Pz ≤ 0.88 as weak; 0.75 ≤ Pz ≤ 0.81 as moderate; 0.67 ≤ Pz ≤
0.74 as strong phospholipase producers.
Antifungal Susceptibility Profile of Candida spp.
Solutions of fluconazole (FLU), itraconazole (ITC), micafungin (MCF), and
amphotericin B (AMB) were prepared in accordance with guidelines M27-A3
(CLSI, 2008a) being diluted in RPMI 1640 (Roswell Park Memorial Institute)
(Angus buffers and Biochemical, Niagara Falls, NY, USA) buffered 3-(N-
morpholino) propanesulfonic acid (MOPS) to pH 7.0. Antifungal drugs tested
were diluted serially in 10 different concentrations, namely: FLU (Pfizer
Incorporated, New York, NY, USA) to 0.125–64 μg/mL; ITC (Pfizer Incorporated,
New York, NY, USA), MCF (Merck, Rahway, NJ, USA); and AMB (Sigma
Chemical Corporation, St. Louis, MO, USA) to 0.015–8 μg/mL. The inoculum of
all strains tested were obtained from 2 h cultivation in SDA at 35°C and an initial
suspension prepared with 90% transmittance determined spectrophotometrically
at 530 nm. Then, two serial dilutions were made, the first in saline solution (1:100)
and the second in RPMI (1:20), in order to obtain final concentration of 103
cells/mL. Susceptibility to antifungal agents was evaluated by broth microdilution,
as recommended within document CLSI M27-A3 (CLSI, 2008a). Aliquots of 100
μL of the final inoculum solution were dispensed in microtiter plates of 96 wells
containing 100 μL of various concentrations of the tested drugs. Finally, the plates
were incubated at 37°C and test reading taken after 24 h incubation for
echinocandins and fluconazole, and after 48h for the other azoles and AMB. Of
note, we have performed readings for voriconazole at 48 h of growth, as
recommended by the document M27-S4 of CLSI when 24 h growth of control is
insufficient (CLSI, 2008b, 2012). All strains were tested in duplicate. MIC was
defined for azoles and echinocandins to the lowest drug concentration which
showed about 50% reduction in turbidity as compared to the positive control well.
For AMB, the MIC was defined as the lowest concentration able to inhibit any
growth visually perceptible (CLSI, 2012).
29
Statistical Analysis
Data were analyzed using the statistical software “Graph Pad, Prism”
version 6.0 and “Stata” version 11.0. Results were presented as mean ± standard
deviation, and differences were analyzed by the One-sample t-test, while the
Spearman coefficient was used to assess the correlation between virulence
factors. For all the analyses, P-values less than 0.05 were considered significant
and the confidence interval of 95% was selected. In addition. all the values
obtained for some of virulence attributes tests in vitro were divided onto tertile
categories as weak, moderate or strong producers.
RESULTS
Microbiological profiling of Candida spp.
Of the 19 samples collected, 190 yeast environmental isolates were
obtained from pigeon dropping collected around the University Hospital. Candida
was the most prevalent genus (83/190, 43.7%), followed by Trichosporon spp.
(44/190, 23.2%), Rhodotorula spp. (43/190, 22.6%), Cryptococcus spp. (13/190,
6.8%) and other genera (7/190, 3.7%). Therefore, we decided to follow up this
investigation including the characterization of the attributes of virulence in vitro
only with 60 Candida spp. strains of medical interest as follows: C. parapsilosis
complex (24/12.6 %), C. tropicalis (19/10.0 %), C. krusei (7 /9.0 %), C. glabrata
(4/2.1 %) and C. rugosa (1/0.5 %; Table 1).
Adherence of Candida spp. to human buccal epithelial cells (HBEC)
All the strains were able to adhere to HBEC. However, the isolates showed
variable expression of this specific virulence factor in vitro (Table 2). C. tropicalis
showed a remarkable ability to adhere to the buccal epithelia. These data can be
observed by both comparing the strains of this species with reference strain
ATCC13803 and when the average values of adhesion for all the strains of this
species were compared to the strains belonging to all the other Candida spp.
evaluated (P < 0.05; Tables 2 and 3). Tertile analysis revealed that most of the
30
isolates of the other Candida spp. showed low ability to adhere to HBEC. In
addition, most of them were also statistically significant more adherent than the
reference strains of each species (Table 2). Strains belonging to C. parapsilosis
species complex were generally statistically more adherent than those belonging
to C. krusei (Table 3). When we evaluated each species separately, for the C.
parapsilosis species complex, LMMM525 was the lowest adherent strain (19 ± 2
Candida cells/ 150 HBEC), while LMMM348 was the strain most adherent (274 ±
2 Candida cells/ 150 HBEC; Table 2). As previously mentioned, C. tropicalis was
represented by highly adherent strains, including the most adherent isolate of the
study (LMMM496; 487 ± 2 Candida cells/ 150 HBEC). The lowest value for
adhesion of this species was found to the strain LMMM312 (75 ± 1 Candida cells/
150 HBEC). Tertile analysis of strains belonging to C. krusei revealed low
adhesion to HBEC, with number ranging from 27 ± 1 Candida cells/ 150 HBEC
(LMMM503) to 91 ± 2 Candida cells/ 150 HBEC (LMMM493). C. glabrata also
showed limited adhesion capacity, (53 ± 4 Candida cells/ 150 HBEC (LMMM174)
to 58 ± 4 Candida cells/ 150 HBEC (LMMM327). The only isolate of C. rugosa
was considered a weak adherent strain.
Evaluation of biofilm formation in Candida spp.
Approximately half of the isolates of the present study was able to form
biofilm on polystirene microtiter plates, while 28 isolates were considered
negative. In addition, several strains belonging to the C. parapsilosis species
complex and C. tropicalis were not able to produce biofilm. Even so, most of the
biofilm forming isolates of C. tropicalis together with C. krusei were considered
either moderate or strong biofilm producers, while C. glabrata and C. parapsilosis
species complex strains showed weak biofilm formation (Table 2). This data was
reinforced with average values comparisons, which showed statistically
significant different results between C. tropicalis and C. krusei strains with the
other Candida spp. (Table 3). For C. parapsilosis species complex strains, the
OD readings ranged from OD570nm of 0.07 ± 0.00 (LMMM346, LMMM513,
LMMM530) to OD570nm of 0.19 ± 0.03 (LMMM527). C. tropicalis reading ranges
were as follows: OD570nm of 0.16 ±0.04 (LMMM179) to OD570nm of 0.59 ± 0.03
(LMMM331). Tertile analysis showed that C. krusei showed very variable levels
of biofilm formation, with the lower biofilm producing strain showing an OD570nm
31
of 0.1 ± 0.01 (LMMM503), whereas the higher biofilm producer strain, showed a
remarkable optical density of OD570nm of 0.8 ± 0.01(LMMM212). C. glabrata
biofilm formation ranged from OD570nm 0.06 ± 0.00 (LMMM180) to OD570nm
0.14 ± 0.00 (LMMM174). The only strain represented by C. rugosa was
considered a strong biofilm producer, according to tertile analysis (Table 2).
Determination of production of proteinase in Candida spp.
From the 61 isolates included in the present study, only 11 (18%) isolates
did not produce the enzyme. The average value for the OD280nm/OD260nm for
all the 61 isolates tested was of 0.05 ± 0.00. Of note, OD readings lower than
0.02 were considered negative, because they represent an amount below the
limit of detection of this technique. The lower amount of proteinase activity was
OD280nm/ OD260nm of 0.02± 0.00 (LMMM503), while the higher level was
OD280nm/OD260nm of 0.38 ± 0.00 (LMMM112), respectively, both from C.
krusei (Table 2). This species, together with C. parapsilosis species complex had
the strongest proteinase producer strains (Table 2). This result was also
confirmed with the average values obtained for all the strains of each species,
except when this comparison was performed between the two previously
mentioned strains and C. glabrata (Table 3). Sixty nine percent of C. parapsilosis
species complex isolates produced more proteinase than the reference strain
ATCC22019 (OD280nm/ OD260nm of 0.03 ± 0.00 (Table 2). Most C. tropicalis
isolates had lower proteinase activity than the reference strain C. tropicalis
ATCC13803, while the opposite happened for the C. rugosa control strain (Table
1). Tertile analysis classified most of the isolates of C. parapsilosis complex as
moderate and strong producers, while isolates of C. tropicalis, C. krusei, C.
glabrata and C. rugosa were classified as moderate and weak producers of the
enzyme. The unique C. rugosa strain produced the following amount of
proteinase: OD280nm/OD600nm of 0.05 ± 0.00 (Table 2).
Determination of production of phospholipase in Candida spp.
Phospholipase production was detected in 95.1% of the isolates when all
the Candida spp. were evaluated. Besides, the levels of enzyme production
varied among isolates of each species. Most C. parapsilosis species complex
strains were considered weak to moderate phospholipase producers (Table 1),
32
while C. krusei and C. glabrata were mostly classified as strong producers (Table
2). Average values comparisons revealed that this difference was significant
between these two and the other previously mentioned species and between C.
tropicalis and the C. parapsilosis species complex (Table 3). Once again, the
amount of secretion of this enzyme was generally lower for the reference strains.
For the Candida parapsilosis species complex, 61% of the strains produced low
levels of phospholipase (14 out of 23 isolates), but the isolate LMMM348 stands
out for showing a high production of the enzyme (0.49 ± 0.03, Table 2).
Determination of production of hemolysins by Candida spp.
Most of strains in the present study were able to produce moderate do high
beta hemolysis on sheep blood agar (59,96,7%). According to Linhares et al.
(2007) classification, 32 isolates (52.4%) presented strong hemolytic activity (HI
≤ 0.43) which is inversely proportional to the HI, while only seven of them (11.4
%) showed low production (HI ≥ 0.56; Table 2). The environmental isolates also
produced equal or above levels of hemolysins than the reference strains of each
species. We could detect only two isolates that did not produce the referred
enzyme (LMMM346 and LMMM348, both belonging to the C. parapsilosis
species complex). Of note, when all the isolates were analyzed together, C.
glabrata strains average values for the HI was statistically significant lower than
the HI found for the others species (meaning high hemolytic activity) and a
statistically significant difference was also observed between strains belonging to
the C. parapsilosis species complex and C. tropicalis (Table 3). In C. parapsilosis
species complex strains the hemolytic activity ranged from 0.34 ± 0.01
(LMMM524) to 0.60 ± 0.04 (LMMM522) and 10 isolates of C. parapsilosis were
strong hemolysins producers (Table 2). C. tropicalis hemolytic activity ranged
from 0.23 ± 0.00 (LMMM496) to 0.65 ± 0.04 (LMMM518) and all C. glabrata
isolates showed lower HI than the reference strain ATCC2001. Interestingly, the
single isolate of C. rugosa (LMMM 216) had a moderate hemolytic enzyme
production, while its reference strain C. rugosa ATCC10571 did not show the
produce the enzyme.
Evaluation of antifungal susceptibility profiling of Candida spp. by the
broth microdilution method
33
All MIC values obtained by the control microorganisms were compatible with
the values expected by the CLSI methodology (2012), ensuring the reliability of
the results obtained for the isolates tested. We observed that all Candida spp.
isolates were susceptible to amphotericin B and Micafungin (Table 4).
Regarding the susceptibility profiling to fluconazole, all the environmental
isolates of the C. parapsilosis complex were susceptible to this drug. However,
only 2 out 19 isolates of C. tropicalis, (LMMM 325, LMMM328; 10.5%) were
considered susceptible to this antifungal drug, while 17 of them (89.5%) were
considered resistant with MICs higher than 64 μg/mL. In addition, all isolates of
C. krusei were considered resistant (as expected), where 12 strains had MIC ≥
64 μg/mL, while for two of them the MIC ≥ 8 μg/mL. For C. glabrata, two isolates
were resistant to this drug, whereas the isolate LMMM327 was considered
susceptible. The single isolate of C. rugosa was susceptible to fluconazole (Table
4).
Regarding the susceptibility profiling to itraconazole, all the isolates
belonging to the C. parapsilosis species complex and C. rugosa were considered
susceptible to this antifungal drug. However, only a single isolate of C. tropicalis
was susceptible to this drug (5.3%; LMMM325), while 94.7% of isolates were
resistant (MIC = ≥64μg / ml). Regarding the isolates of C. krusei, the isolate
LMMM314 showed resistance against itraconazole (MIC= ≥1μg/mL) and the
isolate LMMM338 was susceptible dose-dependent (SDD; MIC= 0.25 μg/mL),
while all the others were susceptible. Two isolates of C. glabrata were susceptible
(MIC≤0.125), while the isolate LMMM327 was considered SDD (MIC= 0.25 μg
/mL).
Discussion
Most of the studies regarding yeasts isolation from pigeon excreta are
focused on Cryptococcus. In our study, Candida spp. together with Trichosporon
spp. and Rhodotorula spp. were more frequently isolated than Cryptococcus spp.
from pigeon droppings. This fact reinforces the potential role of pigeons as a
reservoir of other opportunistic yeasts previously isolated from human infections.
Actually, a few other studies have obtained similar results. For instance, Jang et
al. (2011), found C. glabrata and other Candida spp. (including C. albicans) more
34
frequently than Cryptococcus spp. in a study performed in Seoul, Korea. In the
study of Costa et al., 2010 performed in Northeast Brazil (the same region of the
present study), Candida spp. were also the more frequently isolated species,
mainly C. albicans. Rhodotorula spp. and Trichosporon spp. were also recovered
from the samples in a lower proportion.
In this study, it is possible to observe that Candida spp. strains were able to
express the following virulence factors (in vitro): adhesion to HBEC, biofilm
formation (most, but not all the strains) and the secretion of lytic enzymes, such
as hemolysins, proteinases and phospholipases (Table 2).
In general, the isolates evaluated in this study had a variable capacity of
adhesion to HBECs. A study performed by Modrzwska & Kurn (2015),
emphasizes that adhesion to the surface of host cells is variable according to the
different Candida species. The strains identified as C. tropicalis were
characterized by high adhesion capacity. This species is recognized as highly
adherent to mammalian epithelial and endothelial cells, only preceded by C.
albicans. This phenomenon has been widely described with C. tropicalis clinical
isolates studies from different anatomic sources, including the ones performed in
our group (Chaves et al., 2013). In fact, C. tropicalis shares adhesins also present
in C. albicans, including Alsp (‘Agglutinin-like sequence; (Punithavathy and
Menon, 2012 and Hwp1p (“Hyphal wall protein”; Wan Harun et al. 2013) which
may justify similar high adherence for both species.
In the same manner we found in our study, C. parapsilosis clinical isolates
show moderate adhesion to epithelial cells, while C. glabrata and C. krusei are
weakly adherent. Regarding C. glabrata, it is speculated that the reduced
adhesion capacity of this species results from a lack of adhesins that occur in C.
albicans (Silva et al., 2013; Wan Harun et al. 2013). Our findings suggest that the
strains obtained from fungal droppings behave similarly to clinical isolates, when
adhesion to epithelial cells is investigated.
We also found a remarkable variability to form biofilms in polystyrene
microplates for the strains of all species evaluated. C. krusei was the species with
the highest biofilm formation followed by C. tropicalis. Several studies have
35
demonstrated that C. tropicalis strains obtained from different anatomical sites
and clinical conditions are strongly biofilm producers (Chin et al., 2013).
Data from the literature on biofilm formation of other Candida species, such
as C. parapsilosis, C. krusei and C. glabrata, are very variable, since some
authors report these species as highly biofilm producing, others describe them as
low producers of biofilms. It is noteworthy that the great variability on biofilm
formation is probably related the methodology employed, including the culture
medium, incubation time, plastic material and staining. However, our C. krusei
strains were markedly strong biofilm producers, which is relatively uncommon
among clinical isolates (Treviño-Rangel et al., 2018). Nevertheless, a recent
study proposed that this species may form a well-defined cellular community
biofilm on a stainless steel, simulating the hydrodynamic strength that they are
exposed during food processing in industry, proving that environmental strains of
this species may form a strong biofilm (Brugnonia et al., 2012).
The only isolate of C. rugosa showed a strong biofilm formation.
Interestingly a study with six isolates of C. rugosa obtained from blood, urine,
bronchoalveolar lavage and vaginal secretion has shown that the isolates are
strong biofilm producers (Biasoli et al., 2010) corroborating our results.
Contrary to all expectations, C. krusei showed proteinase production,
despite this species does not have in its genome the SAP gene family (Parra-
Ortega, et al., 2009). However, recently a study of strains from vulvovaginal
candidiasis in Iran reported the production of proteinase by isolates of this
species (Shirkhani et al., 2016). Therefore, other proteinsases (rather than Saps)
mat be present in theis species.
Comparing the proteinase activity among the species, C. krusei, C. glabrata
and C. parapsilosis showed no significant difference between them. The isolates
of the C. parapsilosis complex showed a moderate to strong proteolytic activity,
whereas C. tropicalis isolates were classified as weak producers. The data
obtained in the present study agree with those found by Chaves et al (2013) in a
study carried out with clinical strains of kidney transplant recipients with oral
candidiasis, from the same area of study.
36
Several isolates of the present study were able to produce phospholipase,
being mostly classified as moderate to strong producers. This is contradictory to
several studies that only report the production of this enzyme by C. albicans
clinical isolates (Tellapagrada et al (2014). Actually, Chin et al. (2013) reported
that C. parapsilosis and C. glabrata strains isolated from blood cultures did not
produce the enzyme. Interestingly, our C. glabrata strains showed strong
phospholipase production. In fact, Ghannoum (2000) reported a positive
correlation between C. glabrata isolates that showed phospholipase activity and
remained in the blood despite therapy with antifungal agents and removal of all
intravenous catheters and persistent candidemia. Therefore, such ability to
secrete this enzyme may be an important attribute of virulence produced by our
pigeon dropping strains.
Although some studies claim that the production of hemolysins is only
observed in C. albicans (Negri et al., 2010b). Luo et al., (2001) reported that
NCAC species may produce this enzyme. These data confirm our findings, since
most isolates were able to produce hemolysins.
The highest hemolytic activity occurred in C. glabrata isolates, followed by
C. tropicalis. Our results corroborate with the findings of Riceto et al., (2015) in a
study that evaluated hemolytic activity of different Candida species, which
described a remarkable hemolytic activity for C. glabrata and C. tropicalis
species. In a recent study performed by our group, we were able to demonstrate
high hemolysin activity in C. tropicalis isolated from environmental sources (Zuza-
Alves et al., 2017) corroborating with our findings.
The isolates of C. krusei and C. parapsilosis complex showed moderate
hemolytic activity being contradictory to the findings of Pakshir et al., (2013) with
Candida species isolated from onychomycosis and buccal mucosa, where most
of the isolates of C. parapsilosis did not produce the enzyme. The only isolate of
C. rugosa showed high hemolytic activity, however there are no studies in the
literature to compare this result, reinforcing the novelty of our findings.
We find a considerable number of strains resistant to fluconazole and
itraconazole, specifically in C. tropicalis. Of note, C. krusei is intrinsically resistant
to fluconazole (Silva et al., 2012). Therefore, our results were expected. Although
37
most isolates of C. tropicalis obtained from human patients are susceptible to
azoles, some studies have described the occurrence of resistance in some
clinical isolates of this species to fluconazole (Guinea, 2014), corroborating with
the present study. High Fluconazole resistance was observed among C. tropicalis
strains isolated from beach sand in Brazil (Zuza-Alves et al., 2017). Fluconazole
and Itraconazole cross-resistance have also been previously reported in clinical
(Jiang et al., 2013) and environmental investigations (Zuza-Alves et al., 2017).
Of note, C. glabrata may frequently develop acquired resistance to
fluconazole (Silva et al., 2012) and cross-resistance between this drug and
itraconazole have also been previously reported (Denardi et al., 2015).
Conclusion
In conclusion we may observe the importance of non-Cryptococcus yeasts
isolated from pigeon droppings. The Candida genus was the most prevalent in
our study, with several medically important species being isolated. Of note, these
strains were able to express several virulence factors in vitro, clearly showing
their pathogenic potential. Most importantly, quite a few numbers of strains were
resistant to the azole antifungal drugs without any probable previous explosion to
these antifungal compounds. These findings, together with the fact that they were
collected from the outside of the most important University Hospital in our city,
where air conditioners were present and may throw contaminated pigeon
droppings aerosols into the wards, may be important for environment and patients
colonization and further infection.
Acknowledgements
We are very grateful to Professor Arnaldo Lopes Colombo for the donation
of Candida spp. reference strains.
Competing interests
The authors declare no competing interests
Funding
38
This work was supported by the Federal University of Rio Grande do Norte
Pro-rector of research and postgraduate courses.
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TABLES
Table 1. Prevalence of yeasts obtained from pigeon droppings samples
from the surrounding areas of a tertiaty hospital of the city of Natal, Rio Grande
do Norte state, Northeast region, Brazil.
Table 2. Virulence factors of Candida spp. isolates from the surrounding
areas of a tertiaty hospital of the city of Natal, Rio Grande do Norte state,
Northeast region, Brazil.
Table 3. Average virulence factors comparisons of Candida spp. isolates
from the surrounding areas of a tertiaty hospital of the city of Natal, Rio Grande
do Norte state, Northeast region, Brazil.
Table 4. Results of antifungal susceptibility test of Candida spp. isolates
from the surrounding areas of a tertiaty hospital of the city of Natal, Rio Grande
do Norte state, Northeast region, Brazil.
43
6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES
A etapa inicial do presente estudo teve início na graduação como projeto
de iniciação científica dando continuidade aprofundando os ensaios no
mestrado. A etapa inicial do mestrado foi iniciada com a avaliação dos
fatores de virulência. Essa etapa consumiu um bom tempo, pois os
ensaios eram complexos e como trabalhamos com microrganismos, estes
exigem um cuidado maior no manuseio, que vai desde o cultivo ao
armazenamento.
Os dados iniciais obtidos, desde a iniciação cimentícia, geraram vários
trabalhos científicos, apresentados no 27° Congresso Brasileiro de
Microbiologia, 2013 e no 28° Congresso Brasileiro de Microbiologia, 2015,
Florianópolis.
A extração do DNA demorou um pouco a ser concluída, pois dependia da
disponibilidade de laboratórios parceiros.
A identificação fenotípica realizada por MALDI-TOF não pôde ser
concluída, porque os resultados foram inconclusivos provavelmente por
se tratarem de cepas ambientais, o que acabou atrasando mais ainda o
tempo de defesa.
Como parte do cronograma proposto, a etapa final foi a avaliação da
susceptibilidade dos isolados frente a Anfotericina B, Fluconazol,
Itraconazol e Micafungina, revelando resultados interessantes em cepas
que, provavelmente, não haviam sido expostas a antifúngicos.
44
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PA: Clinical and Laboratory Standards Institute.
19. CLSI, 2008b. M27-S3: Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts; Approved Standard—3rd Informational
Supplement. Wayne, PA: Clinical and Laboratory Standards Institute.
46
20. CLSI, 2012. M27-S4: Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts; Approved Standard—3rd Informational
Supplement. Wayne, PA: Clinical and Laboratory Standards Institut
8. APÊNDICE
Table 1.
Genus or species of yeast Number of enviromental strains (n)
C. parapsilosis species complex 24 (12.6 %)
C. tropicalis 19 (10.0 %)
C. krusei 17 (9.0 %)
C. glabrata 4 (2.1 %)
C. rugosa 1 (0.5 %)
Candida spp. 18 (9.5 %)
Cryptococcus spp. 13 (6.8 %)
Exophiala spp. 3 (1.6 %)
Geotrichum sp. 1 (0.5 %)
Rhodotorula spp. 43 (22.6 %)
Sporobolomyces spp. 3 (1.6 %)
47
Trichosporon spp. 44 (23.2 %)
Total 190 (100%)
Table 2.
48
No of
Candida cells
adhered to
150 HBEC
Biofilm
Formation
(O.D.570nm)
Protease
Activity
(O.D.280nm/O.
D.600)
Phospholipase
zone (Pz)
Hemolysis
Index (HI)
C. parapsilosis
ATCC22019
65 ± 3
0.04 ± 0.00
0.03 ± 0.00
0.83 ± 0.00
Negative
LMMM208
94 ± 4* Negative 0.06 ± 0.01* 0.62 ± 0.03
0.41 ± 0.01*
LMMM339 105 ± 4* Negative Negative 0.73 ± 0.06 0.52 ± 0.02*
LMMM346 131 ± 10* 0.07 ± 0.00 0.03 ± 0.01 0.81 ± 0.03 Negative
LMMM347 34 ± 17* Negative 0.09 ± 0.03* 0.69 ± 0.04* 0.39 ± 0.01*
LMMM348 274 ± 2* 0.16 ± 0.00* 0.05 ± 0.01* 0.49 ± 0.03* Negative
LMMM349 143 ± 3* Negative 0.12 ± 0.01* 0.71 ± 0.04* 0.48 ± 0.02*
LMMM494 52 ± 3* Negative 0.13 ± 0.01* 0.77 ± 0.04 0.39 ± 0.03*
LMMM500 59 ± 7 Negative Negative 0.78 ± 0.03 0.53 ± 0.04*
LMMM502 49 ± 15 Negative 0.08 ± 0.00* 0.71 ± 0.04* 0.43 ± 0.06*
LMMM506 32 ± 3* Negative 0.06 ± 0.01* 0.73 ± 0.04 0.40 ± 0.03*
LMMM507 45 ± 3* Negative 0.11 ± 0.01* 0.73 ± 0.04 0.39 ± 0.02*
LMMM508 104 ± 3* Negative 0.08 ± 0.01* 0.73 ± 0.03 0.39 ± 0.02*
LMMM510 50 ± 2 Negative 0.07 ± 0.01* 0.78 ± 0.04 0.38 ± 0.02*
49
LMMM512 26 ± 4* Negative 0.07 ± 0.02* 0.59 ± 0.03* 0.50 ± 0.00*
LMMM513 33 ± 3* 0.07 ± 0.01 0.13 ± 0.00* 0.62 ± 0.03* 0.46 ± 0.01*
LMMM521 68 ± 2* Negative 0.04 ± 0.01 0.69 ± 0.03* 0.46 ± 0.01*
LMMM522 82 ± 3* Negative 0.04 ± 0.01 0.83 ± 0.00 0.60 ± 0.04*
LMMM523 25 ± 2* 0.09 ± 0.01 Negative 0.68 ± 0.04* 0.44 ± 0.01*
LMMM524 208 ± 7* 0.18 ± 0.02* Negative 0.70 ± 0.00* 0.34 ± 0.01*
LMMM525 19 ± 2* Negative 0.03 ± 0.00 0.69 ± 0.03* 0.44 ± 0.01*
LMMM527 23 ± 3* 0.19 ± 0.03* Negative 0.78 ± 0.00 0.43 ± 0.03*
LMMM528 30 ± 3* Negative 0.10 ± 0.00* 0.53 ± 0.00* 0.56 ± 0.01*
LMMM530 93 ± 2* 0.07 ± 0.00 0.07 ± 0.00* 0.71 ± 0.068 0.36 ± 0.01*
C. tropicalis
ATCC13803
32 ± 2 0.07 ± 0.00 0.04 ± 0.00 0.73 ± 0.05 0.44 ± 0.01
LMMM179 262 ± 2* 0.16 ± 0.04* 0.04 ± 0.00 0.70 ± 0.05 0.39 ± 0.01*
LMMM182 193 ± 2* 0.21± 0.04* 0.03 ± 0.00 0.75 ± 0.04 0.43 ± 0.01
LMMM310 103 ± 2* Negative 0.05 ± 0.00 0.73 ± 0.01 0.39 ± 0.02*
LMMM312 75 ± 1* 0.59 ± 0.03* 0.03 ± 0.00 0.68 ± 0.04 0.38 ± 0.01*
LMMM325 95 ± 2* Negative 0.03 ± 0.00 Negative 0.37 ± 0.01*
LMMM328 215 ± 3* Negative 0.07 ± 0.00* Negative 0.35 ± 0.01*
50
LMMM331 420 ± 1* Negative 0.03 ± 0.00 0.73 ± 0.03 0.38 ± 0.04
LMMM332 120 ± 9* 0.09 ± 0.01 0.02 ± 0.01 0.67 ± 0.05 0.37 ± 0.04
LMMM333 123 ± 5* Negative 0.02 ± 0.02 0.73 ± 0.03 0.48 ± 0.01*
LMMM335 128 ± 7* Negative 0.02 ± 0.00 0.71 ± 0.03 0.39 ± 0.04
LMMM337 132 ± 6* Negative 0.04 ± 0.00 0.62 ± 0.00* 0.39 ± 0.02*
LMMM468 127 ± 6* Negative 0.03 ± 0.00 0.66 ± 0.04 0.38 ± 0.00*
LMMM476 151 ± 11* Negative 0.04 ± 0.00 0.55 ± 0.02* 0.35 ± 0.02*
LMMM483 115 ± 8* 0.28 ± 0.01* 0.02 ± 0.00 0.60 ± 0.03* 0.38 ± 0.00*
LMMM489 168 ± 2* 0.26± 0.02* Negative 0.61 ± 0.02* 0.36 ± 0.00*
LMMM495 154 ± 8* 0.2 ± 0.02* 0.05 ± 0.01* 0.57 ± 0.04* 0.36 ± 0.00
LMMM496 487 ± 2* 0.5 ± 0.01* Negative 0.57 ± 0.04* 0.33 ± 0.00*
LMMM509 165 ± 7* Negative Negative 0.68 ± 0.05 0.43 ± 0.04
LMMM518 121 ± 4* Negative 0.02 ± 0.00 0.68 ± 0.05 0.65 ± 0.04*
C. krusei
ATCC6258
35 ± 1 0.11 ± 0.00 0.19 ± 0.03 0.49 ± 0.04 0.48 ± 0.02
LMMM166 32 ± 3* 0.15 ± 0.00 Negative 0.41 ± 0.06 0.48 ± 0.06
LMMM212 27 ± 2 0.8 ± 0.01* 0.38 ± 0.00* 0.44 ± 0.02 0.55 ± 0.02*
LMMM311 30 ± 2 0.29 ± 0.03* 0.04 ± 0.00* 0.38 ± 0.04* 0.47 ± 0.02
51
LMMM313 64 ± 1* 0.41 ± 0.01* 0.05 ± 0.00* 0.45 ± 0.01 0.46 ± 0.01
LMMM314 46 ± 2* 0.35 ± 0.03* 0.03 ± 0.00* 0.44 ± 0.04 0.52 ± 0.02
LMMM315 50 ± 2* 0.21 ± 0.03* 0.06 ± 0.00* 0.51 ± 0.03 0.61 ± 0.01*
LMMM334 47 ± 3* 0.34 ± 0.04* Negative 0.38 ± 0.04* 0.45 ± 0.04
LMMM336 49 ± 5* 0.2 ± 0.03* Negative 0.78 ± 0.04* 0.38 ± 0.02*
LMMM338 38 ± 2 0.18 ± 0.00* 0.04 ± 0.01* 0.39 ± 0.02* 0.44 ± 0.01*
LMMM490 35 ± 4 0.14 ± 0.03* 0.04 ± 0.01* 0.48 ± 0.02 0.55 ± 0.01*
LMMM493 91 ± 2* 0.21 ± 0.00* 0.03 ± 0.00* 0.51 ± 0.02 0.57 ± 0.01*
LMMM497 35 ± 1 0.36 ± 0.01* 0.03 ± 0.00* 0.37 ± 0.03* 0.53 ± 0.00*
LMMM503 27 ± 1 0.1 ± 0.01 0.02 ± 0.00* 0.45 ± 0.03 0.55 ± 0.02*
LMMM505 32 ± 3 0.14 ± 0.01 0.14 ± 0.01* 0.40 ± 0.03* 0.49 ± 0.03
C. glabrata
ATCC2001
54 ± 6 0.07 ± 0.00 0.05 ± 0.02* 0.52 ± 0.05 0.41 ± 0.01
LMMM174 54 ± 4 0.14 ± 0.00* 0.04 ± 0.01* 0.33 ± 0.02* 0.33 ± 0.03*
LMMM180 53 ± 2 0.06 ± 0.00 0.05 ± 0.00* 0.36 ± 0.04* 0.39 ± 0.02
LMMM327 58 ± 4 Negative 0.10 ± 0.01* 0.46 ± 0.00 0.38 ± 0.01*
C. rugosa
ATCC10571
78 ± 5 0.34 ± 0.00 0.02 ± 0.00* Negative Negative
52
LMMM216 30 ± 2* 0.61 ± 0.00* 0.05 ± 0.00* Negative 0.46 ± 0.02*
* P ˂ 0,05 through Student t
** Not Tested
Gray dark dashed borders stand for strong production, whereas light grey means moderate
production. Non-colored numbers mean weak or negative expression of virulence factors.
53
Table 3.
54
No of Candida
cells adhered to
150 HBEC
Biofilm
Formation
(O.D.570nm)
Protease
Activity
(O.D.280nm/O.D
.600)
Phospholipase
zone (Pz)
Hemolysis
Index (HI)
C. parapsilosis x
C. tropicalis
77 ± 4 versus
176 ± 4*
0.13 ± 0.00
versus
0.26 ± 0.02*
0.06 ± 0.01
versus
0.03 ± 0.00*
0.70 ± 0.03
versus
0.5 ± 0.03*
0.49 ± 0.03
versus
0.4 ± 0.02*
C. parapsilosis x
C. krusei
77 ± 4 versus
43 ± 2*
0.13 ± 0.00
versus
0.27 ± 0.02*
0.06 ± 0.01
versus
0.06 ± 0.00
0.70 ± 0.03
versus 0.45 ±
0.03*
0.49 ± 0.03
versus
0.5 ± 0.02
C. parapsilosis x
C. glabrata
77 ± 4 versus
55 ± 3
0.13 ± 0.00
versus
0.1 ± 0.0
0.06 ± 0.01
versus
0.06 ± 0.01
0.70 ± 0.03
versus 0.38 ±
0.02*
0.49 ± 0.03
versus
0.37 ± 0.02*
C. tropicalis x
C. krusei
176 ± 4 versus
43 ± 2*
0.26 ± 0.02
versus
0.27 ± 0.01
0.03 ± 0.00
versus
0.06 ± 0.00*
0.5 ± 0.03
versus
0.45 ± 0.03*
0.4 ± 0.02 versus
0.5 ± 0.02*
C. tropicalis x
C. glabrata
176 ± 4 versus
55 ± 3*
0.26 ± 0.02
versus
0.1 ± 0.00*
0.03 ± 0.00
versus
0.06 ± 0.01*
0.5 ± 0.03
versus
0.38 ± 0.02*
0.4 ± 0.02 versus
0.37 ± 0.02*
55
Table 4.
Isolates
FLU
ITC
MCF
AMB
C. parapsilosis
ATCC22019
1 (S)
≤0.125 (S)
0.125 (S)
0,125 (S)
LMMM208
0.25(S) ≤0.125 (S) ≤0.03 (S) 0.031 (S)
LMMM339 0.25(S) ≤0.125 (S) 0.125 (S) 0.031 (S)
LMMM346 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.031 (S)
LMMM347 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.031 (S)
LMMM348 0.25(S) ≤0.125 (S) 0.06 (S) 0.062 (S)
LMMM349 0.5(S) ≤0.125 (S) 0.06 (S) 0.031 (S)
LMMM494 0.5(S) ≤0.125 (S) 0.06 (S) 0.125 (S)
LMMM500 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.062 (S)
C. krusei x
C. glabrata
43 ± 2 versus
55 ± 3
0.27 ± 0.01
versus
0.1 ± 0.00*
0.06 ± 0.00
versus
0.06 ± 0.01
0.45 ± 0.03
versus
0.38 ± 0.02
0.5 ± 0.02 versus
0.37 ± 0.02*
56
LMMM502 0.5(S) ≤0.125 (S) ≤0.03 (S) 0,125 (S)
LMMM506 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
LMMM507 0.5(S) ≤0.125 (S) 0.06 (S) 0.25 (S)
LMMM508 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
LMMM510 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.5 (S)
LMMM512 0.5(S) ≤0.125 (S) ≤0.03 (S) 0.5 (S)
LMMM513 0.25(S) ≤0.125 (S) ≤0.03 (S) 0.125 (S)
LMMM521 1(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
LMMM522 1(S) ≤0.125 (S) ≤0.03 (S) 0.5 (S)
LMMM523 0.5(S) ≤0.125 (S) ≤0.03 (S) 0.125 (S)
LMMM524 1(S) ≤0.125 (S) ≤0.03 (S) 0.5 (S)
LMMM525 0.5(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
LMMM527 0.5(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
LMMM528 0.5(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
LMMM530 0.5(S) ≤0.125 (S) ≤0.03 (S) 0.25 (S)
C. tropicalis
ATCC13803
0.25 (S) 0.5 (SDD) ≤ 0.03 (S) 0.1 (S)
LMMM179 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
57
LMMM182 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM310 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM312 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM325 0.125 (S) ≤0.125 (S) ≤ 0.03 (S) 0.062 (S)
LMMM328 ≤2(S) ≥ 64 (R) ≤ 0.03 (S) 0.031 (S)
LMMM331 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM332 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.125 (S)
LMMM333 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.125 (S)
LMMM335 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.125 (S)
LMMM337 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM468 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.125 (S)
LMMM476 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM483 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM489 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM495 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM496 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM509 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
LMMM518 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.25 (S)
58
C. krusei ATCC6258 ≥ 64 (R) ≥ 64 (R) ≤ 0.03 (S) 0.062 (S)
LMMM166 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.25 (S)
LMMM212 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
LMMM311 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
LMMM313 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
LMMM314 ≥ 64 (R) ≥1 R ≤ 0.03 (S) 0.5 (S)
LMMM315 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
LMMM334 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
LMMM336 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 1 (S)
LMMM338 ≥ 64 (R) 0.25 (SDD) ≤ 0.03 (S) 1 (S)
LMMM490 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 1 (S)
LMMM493 8 (R) ≤0.125 (S) ≤ 0.03 (S) 0.25 (S)
LMMM497 8 (R) ≤0.125 (S) ≤ 0.03 (S) ≤0.031(S)
LMMM503 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.062 (S)
LMMM505 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.25 (S)
C. glabrata ATCC2001 0,125 (S) ≤0.125 (S) ≤ 0.03 (S) 0.062 (S)
LMMM174 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
LMMM180 ≥ 64 (R) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
59
LMMM327 2 (S) 0.25 (SDD) ≤ 0.03 (S) 0.5 (S)
C. rugosa ATCC10571 ≤0.031 (S) ≤0.125 (S) ≤ 0.03 (S) ≤0.031(S)
LMMM216 ≤0.031 (S) ≤0.125 (S) ≤ 0.03 (S) 0.5 (S)
