MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL
MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO ...
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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 GENOTIPAGEM E PROTEÔMICA DE ISOLADOS AMBIENTAIS DE Candida tropicalis OBTIDOS DO AMBIENTE COSTEIRO DIANA LUZIA ZUZA ALVES NATAL/RN 2020
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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
GENOTIPAGEM E PROTEÔMICA DE ISOLADOS AMBIENTAIS DE Candida
tropicalis OBTIDOS DO AMBIENTE COSTEIRO
DIANA LUZIA ZUZA ALVES
NATAL/RN
2020
i
DIANA LUZIA ZUZA ALVES
GENOTIPAGEM E PROTEÔMICA DE ISOLADOS AMBIENTAIS DE Candida
tropicalis OBTIDOS DO AMBIENTE COSTEIRO
Tese 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
Doutora em Ciências da Saúde.
Orientador: Prof. Dr. Guilherme Maranhão
Chaves
NATAL/RN
2020
ii
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
Silva, Diana Luzia Zuza Alves.
Genotipagem e proteômica de isolados ambientais de Candida tropicalis obtidos do ambiente costeiro / Diana Luzia Zuza Alves
Silva. - 2020.
134f.: il.
Tese (Doutorado em Ciências da Saúde) - Universidade Federal
do Rio Grande do Norte, Centro de Ciências da Saúde, Programa de
Pós-Graduação em Ciências da Saúde. Natal, RN, 2020.
Orientador: Guilherme Maranhão Chaves.
1. Candida tropicalis - Tese. 2. Estresse osmótico - Tese. 3.
Genotipagem - Tese. 4. Análise proteômica - Tese. I. Chaves,
Guilherme Maranhão. II. Título.
RN/UF/BS-CCS CDU 616.934
Elaborado por ANA CRISTINA DA SILVA LOPES - CRB-15/263BBS
iii
MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenadora do Programa de Pós-Graduação em Ciências da Saúde
Profª. Drª. Ana Katherine Gonçalves Silveira
iv
DIANA LUZIA ZUZA ALVES
GENOTIPAGEM E PROTEÔMICA DE ISOLADOS AMBIENTAIS DE Candida
tropicalis OBTIDOS DO AMBIENTE COSTEIRO
Aprovada em 17/06/2020
Banca examinadora:
Presidente: Prof. Dr. Guilherme Maranhão Chaves (UFRN)
Examinador interno: Profª. Dra Ana Katherine Gonçalves Silveira (UFRN)
Examinador interno: Prof. Dra Aldo da Cunha Medeiros (UFRN)
Examinador externo: Prof. Dr. Reginaldo Gonçalves Lima Neto (UFPE)
Examinador externo: Profª. Drª. Raquel de Melo Barbosa (UNINASSAU)
v
DEDICATÓRIA
Esse trabalho é dedicado à Antonio Fernandes Alves Segundo, à Maria Eliene Zuza
Alves e, com eles, à todos os que sonham com uma vida melhor e batalham por isso,
contando somente com o apoio de Deus e muito esforço. É para aqueles que com
determinação, sacrifício e muito estudo vencem a pobreza, a orfandade, a violência,
o preconceito, o sistema desfavorável. É para aqueles que escolhem todos os dias o
que é certo ao invés do que é fácil; aqueles que entendem o peso da responsabilidade
e não recuam. É para aqueles que pagam o preço para ver o melhor se realizar na
vida de seus filhos. Os frutos doravante colhidos foram semeados por essas pessoas
admiráveis, meus pais, minha referência.
vi
AGRADECIMENTOS
À Deus, Soberano de minha vida, meu tudo.
Aos meus pais, Segundo e Eliene, meu irmão, Arthur e minha irmã amiga
Regina, que é também uma mãe para mim; pelo incentivo, apoio e carinho
inestimáveis.
Ao meu esposo Marcos, amigo de todas as horas, pelo companheirismo e
excepcional compreensão.
Ao meu filho Daniel, alegria de minha vida.
A todos os amigos do Laboratório de Micologia Médica e Molecular,
especialmente Plínio, Mariana, Luanda, Ana Patrícia, Luciana, Sayama, Aurélio,
Laysa, Raíça e Neto, que contribuíram diretamente em etapas importantes do meu
trabalho.
A Profª Christina Araújo, Prof. Aldo Cunha de Medeiros, Profª. Keyla
Rocha, Profª. Analy Salles de Azevedo Melo e aos colegas do Laboratório
Especial de Micologia (UNIFESP) pela valiosa colaboração.
Ao meu orientador, Prof. Guilherme Maranhão Chaves, pela oportunidade de
realizar o que tanto gosto, pelo estímulo, pela confiança e paciência na construção
desse trabalho.
Aos membros da banca examinadora, Profª. Ana Katherine Gonçalves
Silveira, Prof. Aldo Cunha de Medeiros, Prof. Reginaldo Gonçalves de Lima Neto
e Profª. Edeltrudes de Oliveira Lima, por aceitarem o convite para participação na
banca e pelas valiosas sugestões e contribuições científicas.
À Universidade Federal do Rio Grande do Norte e ao Programa de Pós-
Graduação em Ciências da Saúde pela infraestrutura e apoio financeiro.
vii
“Porque tu salvas o povo humilde,
mas os olhos altivos, tu os abates.
Porque fazes resplandecer a minha lâmpada;
o SENHOR, meu Deus, derrama luz nas minhas trevas.
Pois contigo desbarato exércitos,
com o meu Deus salto muralhas.
O caminho de Deus é perfeito;
a palavra do SENHOR é provada;
ele é escudo para todos os que nele se refugiam.
Pois quem é Deus, senão o SENHOR?
E quem é rochedo, senão o nosso Deus?”
Salmos 18: 27-31
viii
LISTA DE FIGURAS
ARTIGO 1
Figura 1 Características fenotípicas de Candida tropicalis. (A): Aspecto
brilhante com borda ligeiramente franjada após 48 h de incubação
a 30 °C em ágar Sabouraud dextrose; (B): Colônias com típica cor
azul escura em meio CHROMagar Candida® após 96 h de
incubação a 35 °C; (C): Aspectos micromorfológicos após
incubação em meio YPD contendo 20% de soro fetal bovino (SFB)
por 7 dias a 30 ° C, 400x: blastoconídios em cadeia simples ou
ramificada, hifas verdadeiras e pseudo-hifas
abundantes....................................................................................
106
Figura 2 Árvore filogenética de Candida spp. O gene do RNA ribossômico
do espaçador transcrito interno 1 (ITS1) -5.8S e as sequências
completas do espaçador transcrito interno 2 (ITS2) e seus
números de acesso, foram obtidos no banco de dados Genbank
em https://www.ncbi.nlm.nih.gov. As sequências foram alinhadas
usando o software BioEdit (v7.2.61). Sequências alinhadas foram
usadas para análise filogenética realizada com o software Mega
7.0.26. O método utilizado para a construção da árvore foi a
parcimônia máxima. A estabilidade do filograma foi acessada
através do método de bootstrapping com 1.000 pseudo-
replicações......................................................................................
107
ARTIGO 2
Figura 1 Dendrograma gerado pelo método de agrupamento de pares não
ponderado com médias aritméticas de 2% de tolerância, de 62
isolados ambientais de Candida tropicalis coletados na praia de
Ponta Negra, Natal, Estado do Rio Grande do Norte, Brasil, de
2012 a 2013. Os destaques em cinza claro representam isolados
obtidos na primeira coleta. Os destaques em cinza médio
representam isolados obtidos na estação chuvosa. Os destaques
em cinza escuro representam isolados obtidos a partir do ponto
geográfico 5....................................................................................
115
ix
Figura 2 Dendrograma MSP com distância relativa entre os isolados
exibidas como unidades arbitrárias. Zero indica similaridade
completa e 1,2 indica alta dissimilaridade. O nível de distância
arbitrária de 0,8 foi escolhido para avaliação de agrupamentos
isolados. O dendrograma representa os principais espectros de
62 cepas ambientais de Candida tropicalis coletadas na praia de
Ponta Negra, Natal, Estado do Rio Grande do Norte, Brasil, de
2012 a 2013. Destaques em cinza claro representam isolados
obtidos na primeira coleta. Os destaques em cinza médio
representam isolados obtidos na estação chuvosa. Os destaques
em cinza escuro representam isolados obtidos a partir do ponto
geográfico 5....................................................................................
117
x
LISTA DE TABELAS
ARTIGO 1
Tabela 1 Métodos convencionais usados para identificação laboratorial de
Candida tropicalis..........................................................................
99
Tabela 2 Proteômica e métodos moleculares para identificação e
genotipagem de Candida tropicalis...............................................
100
Tabela 3 Genes envolvidos nos fatores de virulência de Candida
tropicalis.........................................................................................
102
Tabela 4 Modelos de infecção in vivo e in vitro de Candida
tropicalis.........................................................................................
104
Tabela 5 Genes envolvidos nos mecanismos de resistência antifúngicos
em Candida tropicalis....................................................................
105
ARTIGO 2
Tabela 1 Condições climáticas e coordenadas geográficas dos seis pontos
de coleta de areia da praia de Ponta Negra, Rio Grande do Norte,
Brasil..............................................................................................
112
xi
LISTA DE ABREVIATURAS E SÍMBOLOS
®: Marca registrada
ABC: ATP-binding cassete
SIDA: Síndrome da Imunodeficiência Adquirida
ALS: Agglutinin-like sequence
ATCC: American Type Culture Collection
BCR: Biofilm and Cell Wall Regulator
C.: Candida
CDC: Cell Division Cycle
CDR: Candida Rrug Resistance
CLSI: Clinical and Laboratory Standards Intitute
CNCA: Candida não-Candida albicans
CSA 2: Surface Antigen Protein 2
DNA: Deoxyribonucleic acid
EDTA: Ethylenediamine tetraacetic acid
ELISA: Enzyme Linked Immunosorbent Assay
EPS: Exopolysaccharides
ERG: ERGosterol Biosynthesis
ERIC-PCR: Enterobacterial Repetitive Intergenic Consensus Sequence - Polymerase
Chain Reaction
et al.: Colaboradores
EUA: Estados Unidos da América
FBS: Fetal bovine serum
FKS: FK506 Sensitivity
g/Kg: Grama por kilograma
g/l: Grama por litro
GDP: Guanosine-5'-diphosphate
GPI: Glicofosfatidilinositol
GPS: Global Position System
h: Hora
HBEC: Human buccal epitelial cells
HGC: Hypha‐Specific G1 Cyclin‐Related Protein
HLP: Hemolysin-Like Protein
xii
HOG: High Osmolarity Glycerol
HWP: Hyphal Wall Protein
ITS: Internal transcribed spacer
LMMM: Laboratório de Micologia Médica e Molecular
M: Molar
MALDI–TOF/ MS: Matrix Assisted Laser Desorption/Ionization-Time-of-flight mass
spectrometry
MDR: Multidrug Resistance Gene
MFS: Major Facilitator Superfamily
mg/mL: Miligrama por Mililitro
MgCl2: Cloreto de Magnésio
MIC: Minimal inhibitory concentration
min: Minuto
mL/L: Mililitro por Litro
mL: Mililitro
MLST: Multilocus Sequence Typing
mm: Milímetro
mM: Milimolar
MOPS: 3-(N-Morpholino)propane sulfonic acid
MSP: Main Spectra Profile
NaCl: Cloreto de Sódio
NCAC: Non-Candida albicans Candida
ng/μL: Nanograma por Microlitro
NGY: Neopeptone – Yeast Extract - Glucose
nm: Nanômetro
Nº: Número
NRG: Negative Regulator of Glucose-Repressed Genes
ºC: Grau Celsius
OMS: Organização Mundial da Saúde
PBS: Phosphate Buffered Saline
PCR: Polimerase Chain Reaction
pH: Potencial Hidrogeniônico
PHR: PHotoreactivation Repair deficient
PNA-FISH: Peptide nucleic acid fluorescent in situ hybridization
xiii
QS: Quorum Sensing
RAPD: Randomly Amplified Polymorphic DNA
RBT5: Repressed by TUP1 Protein 5
rDNA: Ribosomal Deoxyribonucleic acid
RHO: Ras HOmolog
RN: Rio Grande do Norte
RNA: Ribonucleic acid
rRNA: Ribosomal Ribonucleic acid
SAP: Secreted Aspartyl Proteinase
SDA: Sabouraud dextrose agar
spp.: Espécies
T.A: Temperatura Ambiente
TM: Trade Mark
U/mL: Unidade por Mililitro
UFRN: Universidade Federal do Rio Grande do Norte
UME: Unscheduled Meiotic Gene Expression
UPGMA: Unweighted Pair Group Method using Arithmetic averages
UV: Ultraviolet
VVC: Vulvovaginal candidiasis
WHO: World Health Organization
WOR: White-opaque regulator
YPD: Yeast peptone dextrose
μg/mL: Micrograma por mililitro
μL: Microlitro
μM: Micromolar
xiv
RESUMO
Candida tropicalis é considerada a segunda espécie mais virulenta do gênero
Candida. Dentre os fatores de virulência relacionados à essa espécie, destaca-se forte
produção de biofilme, adesão a células epiteliais bucais humanas, secreção de
enzimas líticas, morfogênese e phenotipic switching. Além disso, C. tropicalis é
osmotolerante e essa característica é importante para sua persistência no ambiente
costeiro, acarretando risco sanitário para a comunidade. O objetivo desse trabalho foi
realizar uma revisão bibliográfica de C. tropicalis, com foco em todos os assuntos
mencionados. Além disso, utilizou-se as técnicas de microssatélite e MALDI-TOF/ MS
para avaliar a variabilidade genotípica e fenotípica de 62 isolados de C. tropicalis
obtidos de diferentes pontos geográficos de uma praia urbana localizada na região
nordeste do Brasil, durante duas estações climáticas diferentes (estações secas e
chuvosas), avaliando-se também a dinâmica populacional dessa espécie no ambiente
costeiro ao longo do ano. Constatou-se uma tendência de que isolados coletados em
um mesmo período do ano fossem agrupados em um mesmo cluster, pelas duas
técnicas, porém apenas 27 cepas (43,5%) foram colocadas no mesmo cluster nos
dendrogramas do microssatélites e no do MALDI-TOF/MS, o que indica uma
correspondência relativamente baixa entre essas duas técnicas de tipagem. Além
disso, observou-se que isolados obtidos em uma mesma coleta foram agrupados em
um mesmo cluster ou em clusters próximos, e que isolados obtidos do mesmo ponto
geográfico são, na maioria dos casos, considerados idênticos ou altamente
relacionados a pelo menos outro isolado, pelas duas técnicas, indicando relativo grau
de relacionamento. Os métodos empregados também demonstraram a
heterogeneidade de C. tropicalis no ambiente costeiro. Cepas altamente relacionadas
foram encontradas em diferentes pontos geográficos de coleta, demonstrando que C.
tropicalis pode se dispersar por longas distâncias. Tendo em vista o uso incipiente de
MALDI-TOF/MS, são necessários mais estudos para consolidar essa técnica como
ferramenta de tipagem de leveduras, quando comparada à técnica de microssatélite,
que já está bem estabelecida como método de genotipagem de Candida spp.
Palavras-chave: Candida tropicalis, estresse osmótico, genotipagem, análise
proteômica.
xv
ABSTRACT
Candida tropicalis is considered the second most virulent species of the genus
Candida. Among the virulence factors related to this species, there is a strong
production of biofilm, adhesion to human oral epithelial cells, secretion of lytic
enzymes, morphogenesis and phenotipic switching. In addition, C. tropicalis is
osmotolerant and this characteristic is important for its persistence in the coastal
environment, causing health risk to the community. The objective of this study was to
carry out a bibliographic review on C. tropicalis, focusing on all the subjects mentioned
previously. In addition, microsatellite and MALDI-TOF/MS techniques were used to
evaluate the genotypic and phenotypic variability of 62 isolates of C. tropicalis obtained
from different geographical sites of an urban beach located in the northeast of Brazil,
during two different climatic seasons (dry and rainy seasons), besides evaluating the
population dynamics of this species in the coastal environment throughout the year.
There was a trend for isolates collected in the same period of the year to be placed in
the same cluster, by both techniques, however only 27 strains (43.5%) were placed in
the same cluster in both the microsatellite and MALDI-TOF/MS, which suggests a
relatively low correspondence between these two typing techniques. Furthermore, it
was observed that isolates obtained from the same collection timepoint were grouped
within the same cluster or in close clusters, and that isolates obtained from the same
geographic site are, in most cases, considered identical or highly related to at least
one other isolate, by both techniques, suggesting genetic relatdness. The methods
employed also demonstrated the heterogeneity of C. tropicalis in the coastal
environment. Highly related strains were found in different geographic collection sites,
demonstrating that C. tropicalis may be dispersed over long distances. In view of the
incipient use of MALDI-TOF/MS, further studies are necessary to consolidate this
technique as a yeast typing tool, when compared to the microsatellite technique, which
is consolidated as a genotyping method for Candida spp.
Keywords: Candida tropicalis, osmotic stress, genotyping, proteomic analysis.
xvi
SUMÁRIO
DEDICATÓRIA iv
AGRADECIMENTOS v
LISTA DE FIGURAS vii
LISTA DE TABELAS ix
LISTA DE ABREVIATURAS E SÍMBOLOS x
RESUMO xiii
ABSTRACT xiv
1 INTRODUÇÃO........................................................................................... 17
2 JUSTIFICATIVA......................................................................................... 21
3 OBJETIVOS............................................................................................... 22
4 MÉTODOS.................................................................................................. 24
4.1 AMOSTRAGEM.................................................................................... 24
4.2 COLETA DE AREIA.............................................................................. 24
4.3 ISOLAMENTO E PURIFICAÇÃO DE Candida tropicalis...................... 25
4.4 IDENTIFICAÇÃO DAS LEVEDURAS DO SOLO.................................. 25
4.4.1 Análise macroscópica das colônias................................................... 26
4.4.2 Análise micromorfológica das leveduras............................................ 26
4.4.3 Análise fisiológica e bioquímica......................................................... 26
4.4.3.1 Testes de assimilação de fontes de carbono e nitrogênio
(Auxanograma)............................................................................................
26
4.4.3.2 Testes de fermentação de fontes de carbono (Zimograma)........... 27
4.5 Manutenção das leveduras................................................................... 28
4.6 Seleção dos isolados............................................................................ 29
4.7Análise de microssatélite....................................................................... 29
4.8 Análise por MALDI TOF MS.................................................................. 30
5 ARTIGOS PRODUZIDOS........................................................................... 31
AN UPDATE ON Candida tropicalis BASED ON BASIC AND CLINICAL
APPROACHES………………………………………………………………….
32
Candida tropicalis GEOGRAPHIC POPULATION STRUCTURE
MAINTENANCE AND DISPERSION IN THE COASTAL ENVIRONMENT
xvii
MAY BE INFLUENCED BY THE CLIMATIC SEASON AND
ANTHROPOGENIC ACTION………………………………………………….
108
6 CONCLUSÕES…………………………………………………………………. 126
7 COMENTÁRIOS, CRÍTICAS E SUGESTÕES……………………………… 127
8 REFERÊNCIAS…………………………………………………………………. 128
17
1 INTRODUÇÃO
A alta incidência de infecções causadas por leveduras do gênero Candida tem se
destacado entre as infecções de caráter oportunista, principalmente as que envolvem
espécies de Candida não-Candida albicans (CNCA). Dentre essas, C. tropicalis destaca-
se com elevada prevalência, sendo considerada a espécie de CNCA mais isolada na
Ásia [1-3] e a segunda ou terceira espécie de Candida mais isolada no Brasil e nos
demais países da América Latina [4].
O expressivo aumento de infecções por essa levedura em todo o mundo evidencia
seu caráter emergente. Vários atributos de patogenicidade estão envolvidos na virulência
de C. tropicalis, que se inicia com a adesão às células hospedeiras, o primeiro passo
para colonização e infecção [5]. Segue-se a transição morfológica de blastoconídio à hifa
(morfogênese), importante na invasão dos tecidos do hospedeiro e posterior
disseminação da infecção [6]. Para auxiliar na invasão dos tecidos hospedeiros, C.
tropicalis ainda secreta várias enzimas líticas, dentre as quais destacam-se as
proteinases aspárticas secretadas (Saps), as hemolisinas e as fosfolipases [7 - 10]. Além
disso, essa levedura é capaz de formar biofilme, estrutura complexa na qual as células
aderem a superfícies bióticas ou abióticas e secretam sobre si mesmas uma densa
substância polimérica extracelular que proporciona maior resistência do microorganismo
ao ataque fagocítico e resistência a fármacos antifúngicos [11 - 12].
Nesse sentido, nos últimos anos a literatura também tem demonstrado o aumento
da ocorrência de cepas de C. tropicalis com sensibilidade aos antifúngicos reduzida e
outras resistentes in vivo e in vitro. Resistência aos azólicos em espécies de Candida já
foi extensivamente reportada, porém também tem crescido o número de casos de
resistência à anfotericina B e às equinocandinas, a primeira nova classe de anfúngicos
que têm como alvo de ação a parede da célula fúngica, bloqueando a β-1,3-D-glucano
18
sintetase [13]. Estudo realizado por Eschenauer et al. (2014) [14] com 185 isolados de
C. tropicalis relata que 1,4% dessas cepas apresentaram resistência à caspofungina,
anidulafungina e micafungina. O uso indiscriminado de fármacos antifúngicos pode
influenciar o surgimento de cepas de C. tropicalis resistentes, com mutações no gene
ERG3 e consequentes efeitos na síntese de ergosterol [15]. Mutações nos genes ERG11
e ERG6 também já foram encontradas em isolados de C. tropicalis com resistência à
anfotericina B. Além disso, tem sido proposto que mutações concomitantes nos genes
ERG11 e ERG3 levam à resistência cruzada entre anfotericina B e azólicos em C.
tropicalis [16 - 17].
Estudo realizado por Yang et al. (2012) [18] realizou teste de susceptibilidade com
cepas oriundas do solo e isolados clínicos de C. tropicalis. Todos os isolados testados
foram sensíveis à anfotericina B; entretanto, evidenciou-se uma redução da
susceptibilidade em algumas cepas tanto para a anfotericina B como para o voriconazol.
Dentre os isolados do solo, grande parte apresentou reduzida susceptibilidade ao
fluconazol, e todos os isolados clínicos avaliados exibiram essa mesma característica.
Os dados desse estudo sugerem a possibilidade de uma associação entre a limitada
susceptibilidade ao fluconazol e voriconazol de isolados clínicos de C. tropicalis e uma
possível exposição anterior a antifúngicos que possam contaminar o ambiente, como é
o caso de componentes de pesticidas utilizados em práticas agrícolas. É de senso
comum que o uso indiscriminado de antifúngicos pode levar a uma pressão seletiva de
micro-organismos com baixa susceptibilidade a estes fármacos [19].
Os autores sugerem a existência de relacionamento genético entre tais cepas com
sensibilidade reduzida aos azólicos e cepas clínicas de C. tropicalis [18], o que aponta
para o risco de contaminação de pacientes hospitalizados ou indivíduos da comunidade
com isolados dessa levedura de baixa sensibilidade aos azólicos, uma vez que sistemas
19
de tratamento de água e descarte de dejetos de maneira inadequada podem constituir
fonte de contaminação do meio ambiente.
A análise da similaridade genética entre diferentes cepas de uma mesma espécie
de micro-organismo patogênico é procedimento fundamental para permitir a investigação
da história natural de infecções, caracterizar suas fontes e mecanismos de transmissão,
monitorar a emergência de cepas resistentes a drogas e auxiliar na definição de surtos
[20]. Nesse sentido, alguns estudos de epidemiologia molecular têm sido desenvolvidos
com espécies de Candida, principalmente por comparação entre diferentes técnicas de
tipagem molecular, visando discriminar subpopulações de uma mesma espécie e
investigar as relações entre elas. Estudo conduzido por Wu et al. (2017) [21] analisou os
padrões de variabilidade genética de C. tropicalis na ilha de Hainan, China, e investigou
a possibilidade de associação entre diferentes genótipos e o padrão de resistência a
antifúngicos, utilizando isolados obtidos da cavidade oral de indivíduos de cidades
diferentes. Os resultados sugeriram que a população de C. tropicalis de Hainan continha
novas e abundantes variações genéticas nos loci analisados por MLST, além de revelar
evidências de compartilhamento de genótipos entre cepas da Ilha e de outras regiões e
múltiplas origens independentes das cepas resistentes ao fluconazol. Os autores,
portanto, destacam o potencial de dispersão a longa distância de C. tropicalis entre
diferentes regiões geográficas, provavelmente através de atividades humanas, incluindo
a importação e exportação de alimentos colonizados. Ressaltam também a necessidade
de mais estudos que aumentem a compreensão dos mecanismos genéticos
relacionados com a origem e distribuição de genótipos resistentes ao fluconazol.
De fato, comparado a C. albicans, há relativamente poucos trabalhos sobre a
epidemiologia molecular de C. tropicalis, e em se tratando de isolados ambientais os
estudos são claramente escassos. Além disso, muitos atributos de virulência são
expressos ou tem sua expressão modulada em resposta a condições de estresse
20
promovidas pelo ambiente [22]. C. tropicalis é capaz de crescer em meio com
concentração acima de 10-15% de cloreto de sódio, o que explica a razão pela qual esta
espécie é muitas vezes isolada a partir de ambientes salinos [23]. A halotolerância
permite a sobrevivência prolongada de C. tropicalis no ecossistema marítimo. Tal fato
pode constituir importante fonte de adoecimento para a população que frequenta as
praias, como também pode levar à adaptação das leveduras a altas concentrações de
outros íons e à luz UV. Todo esse processo pode se refletir em alterações genéticas que
resultem em pressão de seleção [24].
Sendo assim, dada a alta prevalência de C. tropicalis em todas as partes do globo
e sua crescente importância clínica, é fundamental o entendimento dos padrões de
variabilidade genética dessa levedura.
21
2 JUSTIFICATIVA
Com relação aos determinantes de virulência de C. tropicalis, apesar do grande
número de trabalhos publicados com amostras clínicas em diferentes partes do mundo
e do crescente interesse social pelas questões ambientais, não há até o momento
nenhum estudo referente à caracterização fenotípica de isolados ambientais de C.
tropicalis que investigue o potencial de patogenicidade dessa levedura para
frequentadores de ambientes costeiros, onde essa levedura é amplamente encontrada.
O mesmo pode ser dito a respeito dos estudos de resistência antifúngica. A
resistência aos azólicos em isolados clínicos de C. tropicalis tem sido muito relatada [25-
27], contudo, ainda há relativamente poucos estudos referentes à resistência dessa
espécie a outros antifúngicos, como a anfotericina B, apesar de que resistência a esse
fármaco tem aumentado progressivamente [28], e em se tratando de isolados ambientais
estudos com esse foco são praticamente inexistentes.
Em estudo prévio realizado por nosso grupo de pesquisa, observamos que
isolados ambientais desta espécie, oriundos de ambientes costeiros apresentaram altos
níveis de resistência aos azólicos e à anfotericina B, havendo algumas cepas multi-
resistentes a estas duas diferentes classes de fármacos antifúngicos [28]. Esses isolados
também apresentaram alta capacidade de sobreviver em meios de cultura
hiperosmóticos e acentuada expressão de fatores de virulência in vitro.
Além disso, as praias são nichos com abundância de matéria orgânica e
condições climáticas que podem permitir a prolongada permanência de C. tropicalis no
ambiente bem como a geração de variabilidade genética ainda pouco explorada. Sendo
assim, há urgente necessidade de compreender melhor as origens e distribuições
genotípicas dessa levedura nas diferentes regiões geográficas.
22
3 OBJETIVOS
3.1 OBJETIVO GERAL
Realizar uma atualização de C. tropicalis e avaliar a variabilidade genotípica e
fenotípica de 62 isolados de C. tropicalis obtidos de ambiente costeiro do Nordeste do
Brasil.
3.2 OBJETIVOS ESPECÍFICOS
• Fazer uma revisão da literatura corrente sobre C. tropicalis
contemplando os seguintes aspectos:
• Biologia;
• Taxonomia;
• Características genéticas;
• Métodos de identificação e tipagem;
• Fatores de virulência;
• Modelos de infecção in vivo;
• Tipos de infecção envolvidos;
• Susceptibilidade aos antifúngicos e produtos naturais;
• Resposta ao estresse osmótico e aplicações biotecnológicas;
• Realizar coletas ambientais de amostras de areia da praia e água do mar
em seis diferentes pontos da praia de Ponta Negra, Natal-RN, em dois diferentes
períodos do ano (verão e inverno);
• Realizar o isolamento e purificação de leveduras isoladas da água do
mar e areia da Praia de Ponta Negra, Natal-RN;
23
• Realizar a identificação laboratorial das amostras de leveduras
purificadas, utilizando metodologia clássica;
• Caracterizar genotipicamente os isolados por meio da técnica de
microssatélite;
• Realizar análise proteômica por meio da técnica de MALDI-TOF/MS;
• Comparar os dendrogramas gerados pela análise de microssatélite e
pela técnica de MALDI-TOF/MS, avaliando a variabilidade e a distribuição genotípica;
• Obter dados sobre as condições climáticas dos seis pontos de coleta de
areia, em dois períodos sazonais distintos.
24
4 MÉTODOS
4.1 AMOSTRAGEM
O material para análise consistiu em amostras de 50 g areia seca coletada a 15
centímetros de profundidade em relação à superfície. Foram analisadas amostras
coletadas de seis pontos distribuídos ao longo da orla da praia de Ponta Negra, Natal,
RN, sendo que duas coletas foram realizadas no verão (estação seca) e outras duas no
inverno (estação chuvosa).
4.2 COLETA DE AREIA
As amostras foram obtidas de forma asséptica, seguindo rigorosa observação
das Boas Práticas de Laboratório e de Biossegurança. Foram coletadas em março
(estação seca; C1), abril (estação seca; C2) e julho (estação chuvosa; C3) de 2012 e
em julho (estação chuvosa; C4) de 2013, em seis pontos geograficamente diferentes
da praia, selecionados pela maior concentração de pessoas. Os pontos foram
demarcados com auxílio do programa “Google Earth”, por meio do qual foram obtidas
as coordenadas geográficas dos locais desejados. As coordenadas foram
posteriormente utilizadas para a localização desses pontos, com o auxílio de
equipamento de GPS (Global Positioning System; GARMIN e Trex Vista HCx).
As amostras de areia foram coletadas a 15 cm de profundidade em relação à
superfície, com espátula estéril e transferida para coletores universais estéreis com
volume de 80 mL, devidamente etiquetados e mantidos à temperatura ambiente (T.
A.=28 + 2 ºC), sendo transportados ao Laboratório de Micologia Médica e Molecular
25
(LMMM) do Departamento de Análises Clínicas e Toxicológicas da UFRN para o
processamento laboratorial [29].
4.3 ISOLAMENTO E PURIFICAÇÃO DE C. tropicalis
O processamento laboratorial das amostras foi realizado imediatamente após
a coleta. Em condições rigorosas de assepsia, as amostras de areia foram pesadas e
transferidas dos frascos plásticos, com espátula estéril, para Erlenmeyers de 250 mL
de capacidade, previamente esterilizados. As amostras de 50 gramas de areia foram
diluídas em 90 mL de solução salina 0,9% (w/v) esterilizada e homogeneizadas por 1
minuto, em agitador de tubos (vórtex). A partir da suspensão resultante, retirou-se uma
alíquota de 1 mL, posteriormente semeada na superfície de placas de Petri de 155
mm de diâmetro contendo Ágar Sabouraud Dextrose (ASD) com cloranfenicol (100
mg/L), com auxílio de alça de Drigalski. Após o plaqueamento, as placas de Petri
foram incubadas à T.A., por um período de até 15 dias [29].
Para a purificação dos isolados obtidos a partir da semeadura primária das
amostras de areia e água, cada colônia com aspecto macroscópico de levedura
isolada no ASD, foi repicada para placas de Petri de 90 mm de diâmetro contendo o
meio CHROMagar Candida®, sendo semeadas por esgotamento e incubadas a 30ºC
por 72 horas. As colônias de C. tropicalis apresentam coloração azul petróleo [30]. As
colônias que cresceram isoladamente no CHROMagar Candida®, foram repicadas
para placas de Petri de 90 mm de diâmetro contendo ASD + cloranfenicol e incubadas
a 30ºC por 48 horas, para dar sequência às provas de identificação.
4.4 IDENTIFICAÇÃO DAS LEVEDURAS DO SOLO
26
Para a identificação das leveduras foram observadas as características
macroscópicas e micromorfológicas das colônias que foram isoladas.
4.4.1 Análise macroscópica das colônias
A partir do crescimento fúngico no meio para isolamento primário foi realizada
a caracterização macroscópica das colônias obtidas, observando as características
referentes ao aspecto, coloração do verso e do reverso, textura e tamanho [31].
4.4.2 Análise micromorfológica de leveduras
Após observação do crescimento das colônias nas placas de Petri contendo
ASD com cloranfenicol, foi realizado o microcultivo de cada colônia. Cada amostra foi
semeada com o auxílio de alça em anel de níquel-cromo em três estrias paralelas
sobre a placa de Petri de 90 mm de diâmetro contendo ágar fubá com Tween-80. As
estrias realizadas sobre o ágar foram cobertas com lamínulas estéreis e a placa
incubada à T.A., durante 48-96h. As leituras foram realizadas diariamente, em
microscopia de luz com 400x de magnificação (Olympus, CX21) [31].
4.4.3 Análise fisiológica e bioquímica
4.4.3.1 Testes de assimilação de fontes de carbono e nitrogênio
(Auxanograma)
Para identificação das leveduras em nível de espécie foi utilizado o meio básico
C, para o teste de assimilação de fontes de carbono, e o meio básico N, para o teste
27
de assimilação de fontes de nitrogênio. Para realização dos mesmos, inicialmente foi
preparada uma suspensão com a levedura obtida de dois repiques com 48 horas de
incubação a 30 ºC em placa de Petri contendo ASD com cloranfenicol. Para a
suspensão, utilizou-se tubos de ensaio com 2 mL de água destilada estéril para o meio
C, e tubos com 1 mL de água estéril para o meio N, sendo o inóculo adicionado a
esses tubos de ensaio com alça de níquel-cromo flambada, de modo a obter uma
suspensão de micro-organismos compatível com o padrão da escala 5 de
MacFarland. Subsequentemente, a suspensão de levedura foi adicionada ao meio de
cultura ainda líquido, à temperatura de 50 ºC (meios básico C e N) e a suspensão
distribuída em placas de Petri de 155 mm de diâmetro.
Seguido à solidificação do meio de cultivo, sobre a superfície do meio básico
C, foram adicionados 14 diferentes fontes de carbono em pequenas concentrações,
em posições previamente marcadas na placa de Petri. As fontes de carbono utilizadas
foram: celobiose (Vetec®), dulcitol (Vetec), galactose (Vetec), glicose (Cinética),
inositol (Vetec), lactose (Queel), maltose (Reagen), manitol (Vetec), melibiose (Vetec),
rafinose (Vetec), ramnose (Vetec), sacarose (Reagen), trealose (Vetec) e xilose
(Reagen).
De modo semelhante, sobre a superfície do meio básico N, foi adicionado
nitrato de potássio (KNO3) (Reagen), como fonte de nitrogênio. Peptona (Himedia) foi
utilizada como controle positivo.
As placas foram incubadas à T.A., por 72 horas. Após esse período observou-
se em quais fontes de nutriente havia presença de halo de crescimento fúngico,
indicando a utilização de fontes de carboidrato e ou/nitrogênio pela via oxidativa [31].
4.4.3.2 Testes de fermentação de fontes de carbono (Zimograma)
28
Para avaliar a capacidade fermentativa de diferentes carboidratos pelas
leveduras, foram utilizados tubos de ensaio contendo tubos de Duhram invertidos. Os
tubos de ensaio continham 6 mL do meio básico para fermentação, com peptona,
extrato de levedura e diferentes açúcares. À semelhança do auxanograma,
primeiramente foi preparada uma suspensão com a levedura obtida de dois repiques
com 48 horas de incubação à T.A. em placa de Petri contendo ASD com cloranfenicol.
Para a suspensão, utilizou-se tubos de ensaio com 2 mL de água destilada estéril. O
inóculo foi adicionado a esses tubos de ensaio com alça de níquel-cromo flambada,
para se obter uma suspensão de leveduras compatível com o padrão da escala 5 de
MacFarland.
Foram utilizados para cada cepa de levedura isolada 7 tubos de ensaio com
tubos de Durham invertidos, sendo que cada um continha uma fonte de carboidrato
distinta. Os carboidratos utilizados foram galactose (Vetec), glicose (Cinética), lactose
(Queel), maltose (Reagen), rafinose (Vetec) e trealose (Vetec).
Adicionou-se 200 µl da suspensão de levedura em cada tubo de ensaio,
seguido de homogeneização e incubação à temperatura ambiente. As leituras foram
realizadas diariamente entre 7 e 21 dias, sendo que considerou-se resultado positivo
quando 1/3 a 3/3 do tubo de Durham estava preenchido com gás, produzido pela
fermentação dos carboidratos. O teste foi considerado negativo quando não foi
observada produção de gás dentro do tubo de Durham [31].
4.5 MANUTENÇÃO DAS LEVEDURAS
Todas as cepas de levedura obtidas foram semeadas em tubos cônicos
(Falcon) contendo 6mL de caldo YPD, com auxílio de alça em anel de níquel-cromo.
Os tubos foram, então, incubados “overnight”, à T.A., 200 rpm (Tecnal, TE-420). Um
29
volume de 800 µL do crescimento fúngico foi adicionado a 200 µL de glicerol em
criotubos de 2mL de volume. Os criotubos foram incubados em freezer a -80°C (Termo
Scientific). As cepas de leveduras congeladas foram reativadas por dois repiques
sucessivos em placas de Petri de 90 mm de diâmetro contendo ágar Sabouraud
Dextrose com Cloranfenicol (100 mg/mL) à T.A., previamente à realização dos
experimentos [31].
4.6 SELEÇÃO DOS ISOLADOS
Foram avaliados 62 isolados de C. tropicalis obtidos da areia da praia de Ponta
Negra, Rio Grande do Norte, Brasil, pertencentes à Coleção de Culturas do
Laboratório de Micologia Médica e Molecular (LMMM), Departamento de Análises
Clínicas e Toxicológicas da Universidade Federal do Rio Grande do Norte. Duas
cepas de referência foram utilizadas como organismos controle em todos os testes, a
saber: Uma cepa ATCC (“American Type Culture Collection”) de C. albicans (90028)
e a cepa de C. tropicalis ATCC 13803.
4.7 ANÁLISE DE MICROSSATÉLITE
Para genotipagem por microssatélite, a extração de DNA total a partir de
culturas de C. tropicalis foi feita utilizando-se okit PrepMan® Ultra de acordo com as
instruções do fabricante (Applied Biosystems) empregando-se posteriormente o
primer M13 em reação de PCR [32]. Os produtos de amplificação foram submetidos a
corrida eletroforética em gel de agarose, subsequentemente corado com solução de
brometo de etídio (USB Corporation) e descorado em água destilada. A
fotodocumentação foi realizada em transiluminador de raios UV (UVP – BioDoc – it
30
TM System) e a análise dos resultados por dendrograma utilizando o programa GEL
COMPAR II versão 4.0 Bionumerics (Applied Maths, Kortrijk, Bélgica), através de
análise de agrupamento, de acordo com a similaridade dos padrões de bandas
obtidos. Para gerar o dendrograma, foi utilizado o método UPGMA (“unweighted pair-
group method using arithmetic averages”), que, baseado na matriz de similaridade,
faz o grupamento par a par das amostras, gerando desta maneira o grupamento em
uma árvore enraizada [32].
4.8 ANÁLISE POR MALDI TOF MS
Para a análise por MALDI TOF MS, as proteínas foram extraídas com ácido
fórmico a partir de culturas de C. tropicalis, de acordo com um protocolo adaptado [33
– 34], e a suspensão correspondente de cada isolado foi imediatamente transferida
para uma placa de leitura (Bruker Daltonics - EUA), seguida da adição de solução
matricial (10 mg / mL de ácido alfa-cian-4-hidroxicinamicina etanol: água: acetonitrila
[1: 1: 1]; Sigma - EUA) com 0,03% de ácido. O passo de cristalização ocorreu à
temperatura ambiente e os isolados foram analisados em triplicata. As leituras de
proteínas foram realizadas com um espectrômetro de massa Microflex LT usando a
ferramenta FlexControl 3.0 (Bruker Daltonics, EUA). Para a aquisição de perfis de
proteínas, consideramos uma faixa de massa de 2.000 a 20.000 Da obtida no modo
linear com 40 tiros de laser de nitrogênio com taxas de velocidade variável que
atingem até 60 Hz por poço. Foram utilizadas seis proteínas ribossômicas de
Escherichia coli para calibração externa de massas proteicas analisadas. A geração
de perfis foi realizada usando os softwares Biotyper 3.0 e Biotyper Real Time
Classification (Bruker Daltonik GmbH).
31
5 ARTIGOS PRODUZIDOS
O Artigo “An Update on Candida tropicalis Based on Basic and Clinical
Approaches” foi publicado no periódico Frontiers in Microbiology (ISSN 1664-302X )
com fator de impacto de 4,259 (2017/2018) e Qualis A1; e o artigo “Candida tropicalis
geographic population structure maintenance and dispersion in the coastal
environment may be influenced by the climatic season and anthropogenic action” foi
publicado no periódico Microbial Pathogenesis (ISSN : 0882-4010) com fator de
impacto de 2,581 (2017/2018) e classificação A3, segundo o novo Qualis referência
da CAPES.
32
AN UPDATE ON Candida tropicalis BASED ON BASIC AND
CLINICAL APPROACHES
Diana Luzia Zuza-Alves1, Walicyranison Plinio Silva-Rocha1, Guilherme Maranhão
Chaves1*.
1 Laboratory of Medical and Molecular Mycology, Department of Clinical and
Toxicological Analyses, Federal University of Rio Grande do Norte, Natal city, RN,
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]
33
Abstract 1
Candida tropicalis has emerged as one of the most important Candida species. It has been 2
widely considered the second most virulent Candida species, only preceded by C. 3
albicans. Besides, this species has been recognized as a very strong biofilm producer, 4
surpassing C. albicans in most of the studies. In addition, it produces a wide range of 5
other virulence factors, including: adhesion to buccal epithelial and endothelial cells; the 6
secretion of lytic enzymes, such as proteinases, phospholipases and hemolysins, bud-to-7
hyphae transition (also called morphogenesis) and the phenomenon called phenotypic 8
switching. This is a species very closely related to C. albicans and has been easily 9
identified with both phenotypic and molecular methods. In addition, no cryptic sibling 10
species were yet described in the literature, what is contradictory to some other medically 11
important Candida species. C. tropicalis is a clinically relevant species and may be the 12
second or third etiological agent of candidemia, specifically in Latin American countries 13
and Asia. Antifungal resistance to the azoles, polyenes and echinocandins has already 14
been described. Apart from all these characteristics, C. tropicalis has been considered an 15
osmotolerant microorganism and this ability to survive to high salt concentration may be 16
important for fungal persistence in saline environments. This physiological characteristic 17
makes this species suitable for use in biotechnology processes. Here we describe an 18
update of C. tropicalis, focusing on all these previously mentioned subjects. 19
Key words: Candida tropicalis, virulence factors, antifungal resistance, phenotypic and 20
molecular identification, update 21
34
1. Introduction 1
In the last decades, medicine advances related to the discovery of several medical devices 2
which seek for a longer survival of patients with several infirmities, such as AIDS, 3
hematological malignancies, cancer and other immunosuppressive diseases promoted a 4
longer lifespan. On the other hand, the number of opportunistic fungal infections 5
increased, mainly the ones caused by the Candida genus (Pincus et al., 2007, Araújo et 6
al., 2017). In this context, Candida tropicalis emerges as one of the most important 7
Candida species in terms of epidemiology and virulence. It is able to produce true hyphae, 8
an exclusive property of Candida albicans and its sibling species Candida dubliniensis. 9
C. tropicalis has also been considered a strong biofilm producer species and is highly 10
adherent to epithelial and endothelial cells (Marcos-Zambrano et al., 2014). In addition, 11
several recent investigations have reported the recovery of C. tropicalis resistant to the 12
antifungal drugs currently available, such as the azoles derivatives, amphotericin B and 13
echinocandins (Choi et al., 2016; Seneviratne et al., 2016). In addition, C. tropicalis has 14
been considered an osmotolerant microorganism and this ability to survive to high salt 15
concentration may be important for fungal persistence in saline environments, 16
contributing to the expression of virulence factors in vitro and resistance to antifungal 17
drugs (Zuza-Alves et al., 2016). This property explains C. tropicalis potential use in 18
biotechnological processes such as the production of xylitol from corn fiber and the 19
ethanol from marine algae (Rao et al., 2006; Ra et al., 2015). 20
2. Biology and taxonomy 21
Candida tropicalis was originally isolated from a patient with fungal bronchitis in 1910 22
and named Oidium tropicale (Castellani, 1912). It is a yeast belonging to the filo 23
Ascomycota, from the Hemiascomycetes class (Blandin et al., 2000), which has a single 24
Order created in 1960 by Kudrjavzev, called Saccharomycetales (Kirk et al., 2001). This 25
monophyletic lineage comprises about 1000 known species, including several yeasts of 26
medical importance such as C. tropicalis (Diezmann et al., 2004). 27
According to Kurtzman and Fell (2011) C. tropicais colonies on Sabouraud Dextrose 28
Agar (SDA) are white to cream, with a creamy texture and smooth appearance and may 29
have slightly wrinkled edges. Therefore, it is indistinguishable from other Candida 30
species. After 7 days of microculture on cornmeal agar containing Tween 80, incubated 31
35
at 25 °C, spherical or ovoid blastoconidia, which may be grouped in pairs or alone, 1
measuring approximately 4-8 × 5-11 μm, pseudohyphae in branched chains, and even 2
true hyphae may be observed (Silva et al., 2012; Figure 1). With respect to the 3
biochemical characteristics, it is known that C. tropicalis is capable of fermenting 4
galactose, sucrose, maltose and trehalose, besides assimilating these and others 5
carbohydrates through the oxidative pathway (Kurtzman and Fell, 2011). 6
3. Genetic characteristic 7
C. tropicalis is a diploid yeast, whose genome was sequenced in 2009 (strain MYA-3404) 8
in a study conducted by Butler et al. (2009). It has a genomic size of 14.5 Mb, containing 9
6,258 genes encoding proteins and a guanine-cytosine content of 33.1%. The number of 10
chromosomes is not known with precision, but Doi et al. reported 12 chromosomes per 11
cell for C. tropicalis (Doi et al., 1992). 12
It has been widely believed that C. tropicalis is an asexual yeast. However, some studies 13
performed recently have reported that mating between diploid cells a and α, generating 14
a/α tetraploid cells may occur (Seerva et al., 2013; Porman et al., 2011; Xie et al., 2012). 15
Such mating is regulated by colony phenotypic switching, where cells change from a 16
white to an opaque state. Seervai et al. (2013) demonstrated that tetraploid strains of C. 17
tropicalis can be induced to undergo parasexual cycle without meiotic reduction. This 18
process results in a or α diploid cells competent for mating, being able to form tetraploid 19
cells, which show chromosomal instability after incubation and return to the diploid state 20
after approximately 240 generations (Seervai et al., 2013). Genetic recombination has 21
also been demonstrated, besides ploidy changes (aneuploidies and polyploidy), affecting 22
cells gene expression and protein production (Morrow and Fraser, 2013). This reduction 23
in ploidy is considered a mechanism of adaptation and may be associated with cell stress 24
(Berman and Hadany, 2012). This adaptive mechanism may also generate karyotype 25
variation within the host, and may be induced by various stressors, such as thermal shock, 26
exposure to UV light and growth in l-sorbose or d-arabinose as the only carbon source 27
(Morrow and Fraser, 2013, Bouchonville et al., 2009, Legrand et al., 2008, Arbor et al., 28
2009). It is important to emphasize again that meiosis occurrence has never been 29
described in C. tropicalis. 30
36
C. tropicalis has greater genetic similarity with C. albicans than the other Candida species 1
of medical interest (Butler et al., 2009), as may be observed in Figure 2. This intimate 2
evolutionary relationship is also evident in phenotypic and biochemical characteristics of 3
both species. Phylogenetically, this pattern of evolution can be explained due to 4
predominant clonal reproduction. However, with recombination events frequent enough 5
to generate a population with similar characteristics (Wu et al., 2014). 6
4. Identification 7
4.1 Conventional methods for Candida tropicalis identification 8
C. tropicalis has been quite reasonably well identified with phenotypic methods until the 9
present moment (Table 1). This is contradictory to some other Candida spp., where 10
molecular identification is mandatory due to the existence of cryptic species. 11
Although the classical methodology is of easy execution, it is very laborious and time-12
consuming making it difficult to be used in microbiology routine laboratories (Table 1; 13
Pincus et al., 2007; Sariguzel et al., 2015). 14
The use of chromogenic media, with different substrates that react with specific enzymes 15
of the main Candida species induce the formation of colonies with different colors and 16
has been used for the presumptive identification of C. tropicalis. They have all been used 17
for the screening of distinct species, besides being used to check the purity of Candida 18
colonies and may be helpful to detect mixed infections. Quite a few number of different 19
chromogenic culture media are currently commercially available for yeasts identification, 20
and they have been successfully used for the initial screening of C. tropicalis colonies 21
(Table 1). 22
Several commercially available kits used for yeasts identification based on carbohydrates 23
used by oxidative pathways have been in the market in order to facilitate the process used 24
for yeasts identification (Table 1). C. tropicalis identification with commercial methods 25
have been performed since 1975; since then, several papers have been published in the 26
literature evaluating the efficiency of this method. In a recent study by Stefaniuk and 27
colaborators), the API ID32C system (bioMérieux) was used for the identification of 124 28
Candida clinical isolates, where 21 C. tropicalis isolates (100% of cases) were accurately 29
identified (Stefaniuk et al., 2016). In a study performed by Alfonso et al., with 240 30
37
isolates of different Candida species, the authors found the accurate identification of 34 1
isolates of C. tropicalis with the API ID 32C system (Alfonso et al., 2010). Gundes et 2
al., compared the efficiency of different commercial methods used in the identification 3
116 yeasts of medical interest, demonstrating the accuracy of 87% (101 out of 116) for 4
API 20C® against 82.7% (96 out of 116) with Candifast® system. However, C. tropicalis 5
was accurately identified in 100% of cases with both methods (Gundes et al., 2001). The 6
AuxaColor™ Kit (Bio-Rad) identification has been shown to be accurate in 63,8-95,2% 7
of cases (Pincus et al., 2007). Recently, in a meta-analysis performed by Posteraro et al. 8
(2015), including a total of 26 studies that evaluated yeasts identification methods, they 9
observed that C. tropicalis was accurately identified in 168 out of 184 cases tested with 10
AuxaColor™ and 55 out of 66 cases by using API ID32C® (Posteraro et al., 2015). 11
Besides semi-automated methods currently available, there are other methods completely 12
automated for yeasts identification (Table 1). Won and collaborators performed a study 13
that compared the efficiency of several medically important yeast species identification 14
with the automated systems Vitek2® and BD Phoenix™. This study included a total of 15
341 isolates, from 49 species and C. tropicalis (36 isolates) was accurately identified in 16
34 cases with BD Phoenix™ System and in 32 occasions with Vitek2® (Won et al., 17
2014). 18
The conventional methods of identification including the classical methods, semi-19
automated and automated systems may not be completely accurate on some cases and 20
may lead to an incomplete identification, needing supplementary tests or even give a 21
wrong identification for some species (Chao et al., 2014; Marcos and Pincus, 2013). 22
Therefore, molecular biology advances are of extreme importance for microorganism’s 23
identification because of the fact they are more accurate, and may reduce costs involving 24
identification during the whole process, resulting in a decreased time for the release of 25
results (Chao et al., 2014; Posteraro et al., 2015). 26
4.2 Molecular methods and proteomics for the identification of Candida tropicalis 27
Recently, the evaluation of the protein profile of each species has been used as the basis 28
for yeasts identification and has been proven as more efficient than the conventional 29
methods (Stefaniuk et al., 2016; Chao et al., 2014; Santos et al., 2011). The protein profile 30
38
by mass spectrophotometry is a simple methodology of easy sample preparation and short 1
time for analysis (Table 2; Keceli et al., 2016). 2
The accurate identification of C. tropicalis by proteomics analysis has been demonstrated 3
in several studies which compared identification methods (Sariguzel et al., 2015; 4
Stefaniuk et al., 2016; Chao et al., 2014; Keceli et al., 2016; Angeletti et al., 2015; Panda 5
et al., 2015). C. tropicalis was accurately identified in 22/22 (100%) (Sow et al., 2015), 6
in 21/21 (100%) (Stefaniuk et al., 2016), in 18/18 (100%) (Angeletti et al., 2015), in 17/17 7
(100%) (Chao et al., 2014), in 13/13 (100%) (Keceli et al., 2016) and in 2/2 (100%) by 8
VITEK-MS (Sariguzel et al., 2015). The system performance of the MALDI Biotyper 9
system also showed satisfactory results for the identification of C. tropicalis, where the 10
accurate identification was found for 21/21 (100%) (Stefaniuk et al., 2016), 17/17 (100%) 11
and in 18/18 (100%) (Angeletti et al., 2015) of cases. 12
Several studies have also been performed to evaluate PNA-FISH performance for 13
different Candida species isolated from different anatomic sites, where conclusive results 14
for C. tropicalis ranged from 96-100% of cases (Table 2; Stone et al., 2013; Calderaro et 15
al., 2014; Gorton et al., 2014; Hall et al., 2012). 16
Although the methods used for microorganism’s identification by using PNA-FISH and 17
protein profile analysis using mass spectrophotometry techniques are accurate and have 18
high sensitivity and specificity, molecular sequencing has been considered the gold 19
standard technique for microorganisms identification recently (Keceli et al., 2016). rDNA 20
ITS region sequencing has been quite satisfactorily used for C. tropicalis identification 21
elsewhere. The main target for yeasts DNA molecular sequencing is the ribosomal 22
(rDNA) region (Pincus et al., 2007). This region contains conserved domains separated 23
by variable regions (the small sub unities 18S and 5.8S, besides the large subunit 26S, 24
while these sub unities are separated by the interespacer regions ITS1 and ITS2) which 25
contain species-specific sequences used as the preferential target for universal primers 26
used of identification (Merseguel et al., 2015; Benedetti et al., 2016; Shi et al., 2015, 27
Table 2). 28
4.3 Candida tropicalis genotyping 29
Genotyping methods have largely been used recently to investigate a genetic correlation 30
of different strains of the same species or even among different species (Table 2). These 31
39
methods may be applied to the investigation of infections caused by similar or identical 1
strains, besides the observation of possible micro-evolution or strains substitution during 2
colonization and infection (Almeida et al., 2015; da Costa et al., 2012). 3
Recently, Almeida and collaborators employed RAPD technique with three different 4
random primers (OPA-18, OPE-18 and P4) to evaluate the genetic variability of 15 5
clinical isolates of C. tropicalis obtained from patients with candiduria (Almeida et al., 6
2015). The analyses of the dendrogram constructed with DNA bands with the best 7
discriminatory power primer (OPA-18) showed 4 well defined clusters (I, II, III and IV), 8
where cluster I and II showed above 90% similarity among them, while clusters III and 9
IV had 70% similarity. 10
Costa et al. (2012) genotyped by RAPD 15 strains of C. tropicalis oral isolates with 11
primers OPA-01, OPA-09, OPB-11, OPE-18 and SEQ-06 (da Costa et al., 2012). OPA-12
01 showed the best discriminatory power, presenting ten distinct patterns for C. tropicalis 13
isolates, with 80% similarity (da Costa et al., 2012). Another study using primers OPE-14
03, RP4-2, OPE-18 and AP50-with 12 catheter tip and urine isolates, obtained 9 different 15
clusters with similarities coefficients (SABs) ranging from 0.8-1.0, where different strains 16
were considered unrelated (if SAB was bellow 0.8), moderately related (SAB 0.8-0.89), 17
highly related (SAB 0.90-0.99) and identical (SAB 1.0) (Marol, 2008). 18
Almeida et al. (2015) typed 15 isolates of C. tropicalis with microsatellites and obtained 19
the presence of 5 different alleles with the marker URA3 and 8 different allelic 20
combinations with the CT14 locus, being this marker considered to have a better 21
discriminatory power than the URA3 locus (Almeida et al., 2015). 22
By evaluating 65 clinical isolates of C. tropicalis obtained from different anatomic sites, 23
Wu et al., used different markers of sequence tandem repeats, as follows: Ctrm1, Ctrm7, 24
Ctrm10, Ctrm12, Ctrm15N, Ctrm21, Ctrm24 and Ctrm28 and selected six loci for 25
population genetic analyses (Ctrm1, Ctrm10, Ctrm12, Ctrm21, Ctrm24 e Ctrm28), 26
obtaining a total of 7 (Ctrm24 e Ctrm28) to 27 (Ctrm1) distinct genotypes (Wu et al., 27
2014). 28
The methodology known as MLST (Multilocus Sequence Typing) was originally 29
described by Maiden and colaborators (Maiden et al., 1998). Therefore, by using MLST, 30
strains from different geographic regions and various anatomic sources may be analyzed 31
40
and compared. Strains maintenance, substitution and multiple colonization may be 1
investigated (Wu et al., 2014; Maiden et al., 1998; Wu et al., 2012; Chen et al., 2009). 2
The first MLST studies on C. tropicalis were performed in 2005, by Tavanti et al. with 3
DNA sequencing of 6 housekeeping genes (ICL1, MDR1, SAPT2, SAPT4, XYR1 and 4
ZWF1α). In this study, 106 isolates of C. tropicalis (104 human clinical isolates and 2 5
from animal origin) were evaluated, where 87 DSTs where obtained, grouped within 3 6
different highly related clades (Tavanti et al., 2005). In the study performed by Wu et al., 7
with 58 strains of C. tropicalis from different anatomic sites by MLST, 52 different DSTs 8
grouped within 6 different clades where obtained (Wu et al., 2012). Therefore, MLST is 9
considered a very robust molecular technique used for typing with high discriminatory 10
power, being widely used to evaluate intra-specific variability for different 11
microorganisms including C. tropicalis (Wu et al., 2012; Chen et al., 2009; Odds and 12
Jacobsen, 2008; Tavanti et al., 2005). 13
5. Virulence factors 14
The ability of yeasts to adhere, infect and cause diseases altogether is defined as a 15
potential of virulence or pathogenicity. According to Cauchie et al. (2017), it was 16
previously believed that species of the Candida genus were passively involved in the 17
process of establishment of infection. However, it is now established that these yeasts 18
play an active role in the infectious process through the action of several virulence factors 19
(Cauchie et al., 2017). 20
5.1 Adhesion to epithelial and endothelial cells 21
Adhesion of blastoconidia to host cells is considered the first step for both colonization 22
and the establishment of Candida infections and involves interactions between fungal 23
cells and host surfaces (Cannon and Chaffin, 2001). It is a complex and multiphase 24
process, including different factors, such as the microorganism involved, the composition 25
of adhesion surfaces and several environmental factors (Silva-Dias et al., 2012). 26
Galán-Ladero et al. (2013) performed a study with 29 C. tropicalis isolates with 27
hydrophobicity potential (Galán-Ladero et al., 2013). The cell wall structure is composed 28
by hydrophobic proteins embedded in a cellular matrix which may favor the initial 29
interaction, because hydrophobic particles tend to attach to a high variety of plastic 30
41
materials and host proteins such as laminin, fibrinogen and fibronectin (Tronchin et al., 1
2008). 2
Genes which codify proteins related to adhesion processes are differentially expressed, 3
accordingly to a variety of hosts and environmental conditions (Verstrepen and Klis, 4
2006; Sohn et al., 2006). Despite the fact that ALS genes (Table 3) are highly involved 5
with adhesion in C. albicans, it has been reported that several Candida species also have 6
the ability to adhere to human buccal and vaginal epithelial cells, besides to the 7
gastrointestinal epithelia of mice and several different plastic materials, motivating 8
studies on adhesion in Non-Candida albicans Candida (NCAC) species (Klotz et al., 9
1983). 10
Punithavathy and Menon (2012) evaluated the presence of ALS genes in 48 isolates of C. 11
tropicalis obtained from HIV-negative and positive patients. The authors found that 12 12
isolates (25%) expressed the ALS1 gene, 24 isolates (50%) expressed ALS2 and 23 of 13
them (48%) showed ALS3 expression (Punithavathy and Menon, 2012). 14
HWP1 (“Hyphal wall protein”) gene codify another important adhesin present on the 15
hyphal cell wall (Table 3). In vitro studies demonstrated the presence of high amounts of 16
Hwp1p at hyphal cell walls, while low amounts are present in blastoconidia (Naglik et 17
al., 2006) and pseudo-hyphae (Snide and Sundstrom, 2006). The HWP1 gene is involved 18
in adhesion to human buccal epithelial cells (HBEC), codifying the first protein needed 19
for biofilm formation (Sundstrom et al., 2002; Nobile et al., 2008). 20
The expression of this adhesin was recently reported for C. tropicalis in a study 21
performed in Malaysia (Wan Harun et al., 2013) which investigated the presence of 22
HWP1 in NCAC species by using mRNA expression. HWP1 mRNA transcription was 23
positively regulated in C. tropicalis, indicating the ability of this species to express this 24
adhesin. This study suggests that HWP1 in C. tropicalis shares an identical sequence with 25
C. albicans. Therefore, this is contradictory with the description of the presence of HWP1 26
only in C. albicans (Ten Cate et al., 2009). 27
In fact, most of the studies report C. albicans as more adherent than other NCAC species, 28
but C. tropicalis is considered the second most adherent species of the Candida genus 29
(Calderone and Gow, 2002; Lyon and de Resende, 2006; Biasoli et al., 2010). For 30
instance, Costa et al., evaluated the ability of adherence of Candida isolates obtained 31
42
from the oral cavity of HIV individuals, patients with candidemia and catheter tips and 1
found C. albicans as the most adherent species (average of 227.5 cells/100 HBEC) while 2
C. tropicalis showed in average 123.5 cells/100 HBEC (Costa et al., 2010). Conversely, 3
another study investigating adhesion by oral isolates of C. albicans and C. tropicalis to 4
laminin and fibronectin detected by ELISA, reported C. tropicalis adhesion significantly 5
higher than what was found for C. albicans (da Costa et al., 2012). 6
More recently, Menezes et al. (2013) evaluated the ability Candida spp. clinical isolates 7
to adhere to glass cover slips (Menezes et al., 2013). They found higher adherence of C. 8
tropicalis than C. albicans and yeasts belonging to the C. parapsilosis complex. A recent 9
study performed in Brazil, with isolates from the oral cavity of kidney transplant 10
recipients also demonstrated high ability of adherence to HBEC by C. tropicalis (Chaves 11
et al., 2013). In this study, while C. albicans isolates showed about 237 cells/150 HBECs 12
in average, an isolate of C. tropicalis had 335 cells/150 HBECs, reinforcing the 13
remarkable role of adhesion as an important virulence factor in C. tropicalis. 14
5.2 Morphogenesis and phenotypic switching 15
Subsequently to the adhesion step to host cells, bud-to-hyphae transition (also called 16
morphogenesis) is highly relevant to some pathogenic yeasts, including Candida spp. 17
(Calderone and Gow, 2002). It is one of the most important steps for the establishment 18
of candidiasis and is considered a necessary step for several virulence processes, 19
including invasion of host epithelial layers, endothelial rupture, survival to phagocytic 20
cells attack, biofilm formation and thigmotropism (Lackey et al., 2013). 21
Studies concerning morphogenesis are very well established for C. albicans, with very 22
well established environmental signals, transcription regulators and target genes involved 23
in filamention (Lackey et al., 2013; Gustin et al., 1998; Kumamoto and Vinces, 2005; 24
Wapinski et al., 2007). However, there are considerably less studies concerning 25
morphogenesis in other NCAC species. Several Candida species may develop pseudo-26
hyphae, but quite a few are able to form true hypahe, including C. albicans, C. 27
dubliniensis and C. tropicalis. The latter do not show the same degree of filamentation 28
than C. albicans; however, because of the fact they are frequently associated with 29
infectious processes, they certainly have mechanisms of adaptation that may favor 30
filamentation in specific environmental conditions (Lackey et al., 2013). 31
43
Galán-Ladero et al. (2013) evaluated the filamentation among C. tropicalis isolates 1
obtained from different anatomic sites of patients admitted in a Spanish tertiary hospital 2
(Galán-Ladero et al., 2013). The authors described high levels of filamentation for 76.6% 3
of the isolates at the specific environmental conditions. Wapinski et al. (2007) reported 4
that at least 55 out of the 105 genes involved in C. albicans filamentation are conserved 5
in C. tropicalis (Wapinski et al., 2007). 6
Lakey et al. (2013) induced C. tropicalis cells filamentation and analyzed gene 7
expression at the conditions provided (Lackey et al., 2013). They found significant 8
filamentation in serum and glucose medium at 37 °C. Optical microscopy showed the 9
presence of elongated yeast-like cells, pseudohyphae and true hyphae that were shorter 10
than the ones found in C. albicans. They also verified that the negatively regulated gene 11
NRG1 has an important role in inhibiting filamentation in other NCAC species, 12
suggesting that this gene may be related with poorer filamentation found among these 13
species (Table 3). The UME6 gene is transcriptionally induced during filamentation in 14
C. tropicalis, similarly to what happens in C. albicans (Table 3; Banerjee et al., 2013) 15
Porman et al .(2013) reported the elevated expression of the transcriptional regulator 16
WOR1 (Table 3) in C. tropicalis cells cultivated on Spider medium (Porman et al., 2013). 17
The micromorphological analysis of isolates with wrinkled phenotype showed that the 18
most filamentous strains had WOR1 overexpression. Wor1p homologues were also 19
found in Saccharomyces cerevisiae (Cain et al., 2012) and Histoplasma capsulatum 20
(Nguyen and Sil, 2008), controlling morphological transition within these species. This 21
finding may suggest the existence of a common ancestor gene found in the C. tropicalis 22
genome (Porman et al., 2013). 23
In addition, Wor1 which is the master regulator of the white-opaque switching, a 24
phenomenon which is related to the reversible transition of cells from a white phase to 25
an opaque phase, where cells are larger and elongated, while colonies have wrinkled 26
appearance (Slutsky et al.,1987). Besides morphology, these two different cell types 27
exhibit dramatic differences regarding to the preferred anatomic sites they colonize and 28
infect, in addition to specific responses to environmental and nutritional signals and 29
mating behavior (Mancera et al., 2015). In C. tropicalis, WOR1 overexpression direct 30
cells to the opaque phase which is involved in biofilm formation and morphogenesis 31
(Porman et al., 2013). 32
44
It was described in the C. tropicalis genome an ortholog of the transcription factor Efg1 1
(enhanced filamentous growth), commonly found in C. albicans (Table 3; Mancera et 2
al., 2015). The deletion of both alleles of the EFG1 gene revealed that Efg1p is essential 3
for filamentation, biofilm formation and white-opaque switching in C. tropicalis, 4
similarly to C. albicans, indicating conservation in the function of this ortholog gene. 5
Zhang et al. (2016) reported a grey phenotype in C. tropicalis recently, whose cells are 6
small and elongated, show intermediate mating competence and virulence in rats’ animal 7
models (Zhang et al., 2016). 8
5.3 Biofilm formation 9
The ability of yeast cells to form biofilms is an important determinant of virulence in 10
Candida spp. and has been considered the main form of microbial growth recently 11
(Donlan and Costerton, 2002; Fanning and Mitchell, 2012). Biofilms are complex 12
structures formed by a community of microorganisms adhered to solid surfaces of either 13
biotic or abiotic nature. Therefore, in vitro biofilm formation may be organized by three 14
important steps, as follows: adhesion and colonization of yeast cells on a surface; cellular 15
growth and proliferation, forming a basal layer; and pseudohyphal and/or true hyphal 16
formation (for the species that are able to form filaments), with the subsequent secretion 17
of an exopolymeric extracellular matrix which embeds microorganisms with low growth 18
rates and altered phenotypes (Hawser and Douglas, 1995; Baillie and Douglas, 1999; 19
Chandra et al., 2001; Ramage et al;. 2001; Douglas, 2003). The exopolymeric matrix 20
(EPS) may be secreted by different populations of either unique or multiple microbial 21
species (Adam et al., 2002). Some advantages of biofilm formation include: the 22
protection of microorganisms against environmental damage, nutrients availability, 23
metabolic cooperation and the acquisition of genetic modification (Douglas, 2002). 24
The formation of the microbial community involves a cascade of molecular mechanisms 25
and fine alterations in gene expression (Nobile and Mitchell, 2006; Araújo et al., 2017). 26
Signaling molecules which naturally occur in fungal cells as a response to environmental 27
stimuli are part of this process present in the Candida genus (Ramage et al., 2006). This 28
regulation is called “quorum sensing” (QS) mechanism and is the main communication 29
form among several microorganisms correlated to population density (Albuquerque and 30
Casadevall, 2012). 31
45
Farnesol is kind of self-regulator, a sesquiterpene with the ability to inhibit biofilm 1
formation and altering the expression of 274 genes in C. albicans, specifically involved 2
in filamentation. Weber et al. (2010) investigated the role of farnesol in biofilm formation 3
of C. tropicalis. They found that besides inhibiting cellular aggregates, cells of the C. 4
tropicalis mature biofilm were also influenced by farnesol, which may be related to their 5
dispersion to other body sites (Ramage et al., 2006; Nickerson et al., 2006). 6
The initial step for biofilm formation is dependent of cellular adhesion cells to substrates 7
and further formation of a basal layer (Nobile and Mitchell, 2006). C. tropicalis adhesins 8
are also involved in biofilm formation (Punithavathy and Menon, 2012; Wan Harum et 9
al., 2013, Table 3), and are regulated by the BCR1 gene (also considered a cell wall 10
regulator). In addition, the RBT5 gene was also found in the C. tropicalis genome 11
(Fitzpatrick et al., 2010). 12
Other genes involved in C. tropicalis biofilm formation are WOR1, UME6, NRG1, 13
ERG11 and MDR1 (Table 3). Besides being involved with morphogenesis and phenotypic 14
switching, WOR1 is one of the main transcriptional factors involved in biofilm formation 15
(Porman et al., 2013; Xie et al., 2012). 16
UME6 and NRG1 are key transcription regulators directly involved in morphogenesis in 17
C. tropicalis (Finkel and Mitchell, 2011). The overexpession of UME6 reduces the 18
liberation of mature sessile cells, while the decreased expression of NRG1 promotes cells 19
dispersion (Uppuluri et al., 2010). 20
With respect to the expression of resistance genes to antifungal drugs, ERG11 (ergosterol 21
biosynthesis) and MDR1 (multidrug resistance) genes (Table 3) are related with 22
resistance to fluconazole. Bizerra et al. (2008) reported the increased expression of these 23
genes in sessile cells of C. tropicalis isolated from vulvovaginal candidiasis (VVC) and 24
uroculture resistant to both fluconazole and amphotericin B. Punithavaty et al.(2012) also 25
demonstrated higher resistance to fluconazole of sessile cells liberated from mature 26
biofilms of C. tropicalis. 27
There are evidences that biofilm cells formed on medical devices constantly released in 28
the bloodstream guarantee the successful establishment of disseminated candidiasis 29
(Fanning and Mitchell, 2012). Marcos-Zambrano et al. (2014) investigated biofilm 30
formation in different Candida species obtained from episodes of fungemia and found C. 31
46
tropicalis isolates were the strongest biofilm producers. In fact, another study reported 1
that the high thickness of the EPS matrix of C. tropicalis biofilm cells may impair oxygen 2
and nutrients diffusion to cells, and may be responsible for the lower metabolic activity 3
(Alnuaimi et al., 2013). 4
Pannanusorn et al. (2013) also described C. tropicalis as the most efficient biofilm 5
producers among bloodstream isolates as compared to other NCAC species. Paiva et al. 6
(2012) evaluated the in vitro biofilm formation by C. tropicalis isolates obtained from 7
VVC. This species was also considered the strongest biofilm producer compared to C. 8
albicans, yeasts belonging to the C. parapsilosis complex, C. glabrata and C. 9
guilliermondii. A similar trend was also described by Udayalaxmi et al. (2014) with 10
strains isolated from the urogenital tract (samples from vaginal fluid and urine) of 11
patients from a tertiary hospital in the South of India. Therefore, C. tropicalis has been 12
considered an important biofilm producer species of the Candida genus. 13
5.4 Lytic enzymes 14
In order to facilitate host tissues invasions, several pathogenic microbes secrete lytic 15
enzymes such as proteinases, phospholipases and hemolysins to destroy, alter or damage 16
the integrity of host membranes, leading to the dysfunction or rupture of host cells (Sanita 17
et al., 2014). 18
Pathogenic Candida species produce a great variety of hydrolases, including secreted 19
aspartic proteinases (Saps). These proteins have been intensely investigated, and possess 20
a wide range of substrates, including collagen, queratin and mucin. They have the ability 21
to degrade epithelial barriers, antibodies, complement and cytokines (Hube and Naglik, 22
2001), and are encoded by a great gene family. The SAP gene family is composed by 10 23
genes and was initially described in C. albicans (Ruchel et al., 1983). These genes are 24
differentially regulated and expressed under several laboratory conditions and are 25
activated during different stages of infections in vivo. In addition, some of the SAP genes 26
are more important to superficial rather than systemic infections, and are also involved in 27
other pathogenic process in C. albicans, such as adhesion, host tissue invasion and 28
immunological system cells evasion (Hube and Naglik, 2001). 29
47
It is well known since 1983 that C. tropicalis is able to secrete proteinases as one of the 1
most important determinants of virulence of this species (Ruchel et al., 1983; Macdonald 2
and Odds, 1983). In 1991, Togni et al., reported the nucleotide sequence of a gene 3
involved with the extracellular secretion of proteinases by this yeast, while in 1996 the 4
same authors reported the secretion of Sapt1p by C. tropicalis (Togni et al., 1996). 5
Subsequently, the crystallographic structure of this protein was published, and was 6
considered very similar to the Sap2p of C. albicans (Symersky et al., 1997). 7
A study performed by Zaugg et al. (2001) suggested the existence of a SAPT gene family 8
in the C. tropicalis genome, leading to 4 genes cloning: SAPT (1-4; Table 3). However, 9
only Sapt1p was purified from culture supernatant and biochemically characterized. 10
Silva et al. (2011) investigated epithelial invasion by C. tropicalis using a reconstituted 11
human buccal epithelia model. All the isolates tested were able to colonize this tissue and 12
cause a great damage after 24 h. Real time PCR showed that SAPT2-4 transcripts were 13
detected, while SAPT1 expression was rarely observed. In addition, the authors showed 14
that there was no increase in SAPT1 expression, suggesting that the high invasive capacity 15
of C. tropicalis may not be related with the specific expression of this gene. Following 16
the same trend, Togni et al. (1996) reported that SAPT1 gene disruption in C. tropicalis 17
seemed to have low effect in attenuation of virulence in mice, in a model of systemic 18
infection. 19
Costa et al.(2010) evaluated proteinase activity of 15 isolates of C. albicans and 15 of C. 20
tropicalis obtained from the saliva of dental patients in Brazil. All C. tropicalis isolates 21
showed higher enzymatic production than C. albicans. These results are contradictory to 22
most of the studies which suggest higher proteinase activity in C. albicans than in C. 23
tropicalis (Zaugg et al., 2001; Sachin et al., 2012). 24
In addition to the secretion of proteinases, the secretion of phospholipases constitutes 25
important determinants of virulence in Candida spp. This heterogeneous group of 26
enzymes catalyzes the hydrolysis of ester bonds in glycerol phospholipids, with each 27
enzyme participating in a specific reaction (Ghannoum, 2000). Secretion of 28
phospholipases is therefore considered a key attribute for invasion of host epithelia, since 29
phospholipids are major components of all cell membranes. In addition, the breakdown 30
48
of these molecules promotes great instability in host cells, resulting in cellular lysis 1
(Schaller et al., 2005). 2
One of the first studies that analyzed the production of phospholipases in Candida spp. 3
was published in 1984 by Samaranayake et al. which demonstrated the secretion of these 4
enzymes only in C. albicans isolates, without any detection in C. tropicalis. However, 5
other authors later reported phospholipase activity in isolates of this species. A recent 6
study conducted by Jiang et al. (2016) with 52 strains of C. tropicalis found 7
phospholipase activity in 31 isolates from different clinical sources. However, strains 8
showed low enzyme production. Another study with 29 strains of several anatomic sites 9
obtained from hospitalized patients, described low or no phospholipase activity in C. 10
tropicalis (Galan-Ladero et al., 2010). 11
Conversely, a study conducted by Deorukhkar et al. (2014) investigating the expression 12
of several virulence factors in 125 clinical isolates of this species concluded that the 13
secretion of phospholipases was the main determinant of virulence expressed by these 14
strains. The authors suggest that the variability of results between different authors may 15
be a result of biological differences among the isolates tested. 16
Related to the expression of these enzymes in the presence of antifungal drugs, Anil and 17
Samaranayake (2003) analyzed the effect of previous exposure of C. albicans and C. 18
tropicalis to antifungal drugs on extracellular phospholipase activity. They concluded that 19
the enzymatic activity of both species reduced significantly after previous exposure to 20
nystatin and amphotericin B. In fact, they showed that C. albicans had greater 21
phospholipase expression than C. tropicalis. 22
Phospholipases are classified into four major groups, named from A to D, all already well 23
described for C. albicans (Schaller et al., 2005). However, a few studies address this gene 24
regulation in C. tropicalis. Phospholipase B (PLB; Table 3) is known to catalyze the 25
hydrolytic cleavage of sn-1 and acyclic glycerophospholipid sn-2 esters (Ghannoum, 26
2000) and is primarily responsible for phospholipase activity in C. albicans (Schaller et 27
al., 2005). 28
In 1998, Hoover et al. published an investigation with degenerate oligonucleotides 29
(derived from conserved regions of the PLB1 gene of Saccharomyces cerevisiae and other 30
fungi) to amplify homologous fragments of PLB1 in C. albicans and C. tropicalis by 31
49
PCR. The main PCR product obtained was a 540 bp fragment with a high probability of 1
PLB1-correspondence of other fungi, and significant homology was found between the 2
deduced amino acid sequence of the PCR product of C. albicans and C. tropicalis and the 3
corresponding regions of PLB1 sequence of S. cerevisiae, Torulaspora delbrueckii and 4
Penicillium notatum (~ 70-75% resemblance, ~ 55-65% identity). In that same year, 5
Bennet et al. (1998) evaluated the presence of homologous sequences to C. albicans PLC 6
in NCAC species, including five isolates of C. tropicalis. A DNA sequence homologous 7
to CAPLC1 was detected in only 3 of these isolates. Thus, the need for further studies 8
addressing the molecular mechanisms related to phospholipase activity in C. tropicalis is 9
evident. 10
The hemolysins are another group of proteins that significantly contribute for the 11
dissemination of Candida infections, specifically in facilitating hyphal penetration in host 12
tissues (Luo et al., 2004; Tsang et al., 2007). Hemolytic factors secreted by fungi cause 13
hemoglobin liberation from red blood cells for further utilization by yeasts as an iron 14
source (Giolo and Svidzinski, 2010). This chemical element is an essential cofactor to a 15
great number of metabolic processes, such as oxygen transport, gene expression 16
regulation and DNA synthesis. Therefore, the ability of iron acquisition is of fundamental 17
importance for microorganisms survival and establishment of infectious processes (Giolo 18
and Svidzinski, 2010). 19
Manns et al. (1994) reported iron acquisition from erythrocytes by C. albicans as a 20
consequence of a protein factor that promoted host cells lysis. In 1997, Tanaka et al., 21
reported that this factor is liberated from the culture medium supernatant, and concluded 22
that it was a cell wall manoprotein. The same phenomenon was observed in C. tropicalis 23
(Favero et al., 2014), and although this factor is known as directly involved with yeasts 24
pathogenicity, it is still poorly understood (Favero et al., 2011). 25
The study conducted by Luo et al. (2001) was the first one to show differences in 26
hemolysin production by different Candida species on SDA plates containing sheep 27
blood. The authors also observed that the hemolysis induced by this method could be 28
divided into categories according to the standard microbiological nomenclature, 29
including: total hemolysis (beta), partial hemolysis (alpha) or hemolysis absence (gama). 30
In this study with 80 isolates of 14 different Candida species, all the 5 isolates of C. 31
tropicalis showed a large clear halo around colonies, proving the ability of C. tropicalis 32
50
in producing beta hemolysis. Similarly, Favero et al. (2011) detected hemolysin 1
production in C. tropicalis strains after incubation in both solid and liquid SDA 2
containing either human or sheep blood. 3
A study produced by Rossoni et al. (2013) evaluated the hemolytic activity in different 4
Candida species obtained from the oral cavity of HIV positive patients. Strong hemolytic 5
activity was observed in 75% of C. tropicalis isolates evaluated, only after C. albicans. 6
Similar results were found for Candida isolates obtained from different anatomical sites 7
(blood, synovial and peritoneal liquid) where, again, C. albicans proceeded C. tropicalis 8
in hemolysins production (de Melo Riceto et al., 2015). 9
Contradictory to these results, Favero et al. (2014) analyzing clinical Candida spp. 10
isolates from bloodstream infection, reported low hemolytic activity in C. albicans, while 11
C. tropicalis was the species tested with greater hemolysins production. 12
The genetic regulation of hemolysins production in the Candida genus was not still 13
largely investigated (Anil et al., 2014). It is known that in C. glabrata, the HLP gene 14
(hemolysin-like protein) encodes a protein associated with hemolytic activity (Luo et al., 15
2004). In C. albicans the Csap is involved with iron acquisition from host erythrocytes 16
during hyphal development (Okamoto-Shibayama et al., 2014). This enzyme is a member 17
of the Rbt5 protein (Table 3), also described in C. tropicalis, as previously mentioned. 18
However, there are currently no studies in the literature concerning the genetic elucidation 19
of hemolytic activity in C. tropicalis. 20
6. In vivo models of infection by Candida tropicalis 21
The characterization of the expression of most variable virulence factors by Candida spp. 22
and other fungi are necessary for the understanding each particular pathway involved in 23
microorganisms pathogenicity (de Campos Rasteiro et al., 2014; Solis and Filler, 2012; 24
Takakura et al., 2003). However, experiments performed in vivo involve different 25
variables which cannot be controlled like what happens in experimental conditions in 26
vitro, including the presence of body fluids, pH variation, commensal microorganisms 27
and their metabolites and host response during infection. Therefore, in vivo experimental 28
models are needed for the global understanding of infectious disease pathogenicity, 29
interactions with host cells and immune response as well as it is a more appropriate 30
51
approach to evaluate new therapeutic strategies (de Campos Rasteiro et al., 2014; Solis 1
and Filler, 2012; Takakura et al., 2003). 2
Several studies have been described in the literature with animal models of infections by 3
C. tropicalis using mice (Bayegan et al., 2010; Chen et al., 2014; Koga-Ito et al., 2011; 4
Mariné et al., 2010; Nash et al., 2016; Wang et al., 2016; Zhang et al., 2016). Nash et al., 5
(2016) evaluated the co-infection of 6 different Candida species (C. albicans, C. 6
tropicalis, C. parapsilosis, C. krusei, C. dubliniensis and C. glabrata) with 7
Staphylococcus aureus, intraperitoneally inoculated. They evaluated mortality rates and 8
attributed a score of 1-4 to evaluate characteristics of morbidity (creepy hair, absence of 9
mobility, arched posture and ocular secretion). C. tropicalis associated with S. aureus 10
showed the second highest mortality rate (behind C. albicans) and a mortality index of 3 11
(Nash et al., 2016). 12
Animal models of systemic infections may be induced by the inoculation of C. tropicalis 13
via the lateral tail vein (Table 4; Zhang et al., 2016). This via of infection was established 14
by Zhang et al., (2016) to evaluate virulence of a new phenotype described by C. 15
tropicalis, the “Grey phenotype”, besides the other phenotypes already described (White–16
Opaque). After systemic infection through the tail vein with strains of each phenotype 17
(Gray, White and Opaque), animal organs have been removed and macerated and fungal 18
load was evaluated. The authors found that cells of the Grey phenotype showed 19
intermediate distribution, but greater than cells with the White phenotype for all the 20
organs evaluated (kidney, lungs, spleen, liver and brain), 24h and 7 days after infection. 21
Other mice models of Candida infections have been described in the literature such as the 22
VVC model described by Fidel et al. (1997), where doses of estradiol valerate are 23
subcutaneously administered (0.1 mg/100µl of sesame oil) in the vagina of animals 24
infected with 5x104 cells/20µl PBS in order to successfully establish the vaginal infection 25
(Fidel et al., 1997; Fidel et al., 1996; Garvey et al., 2015; Nash et al., 2016). Nevertheless, 26
to the best of our knowledge, they were still not employed for the experimental 27
investigation of VVC caused by C. tropicalis. 28
Alternative models of experimental infections have been broadly used for virulence and 29
interactions with the host studies (Table 4; de Souza et al., 2015; Forastiero et al., 2013; 30
Hamamoto et al., 2004; Ishii et al., 2015; Mesa-Arango et al., 2013; Shu et al., 2016; 31
52
Zanette and Kontoyiannis, 2013). Several factors are considered as an advantage for the 1
utilization of a model of infection using larvae, including an easier manipulation and 2
lower maintenance cost (de Souza et al., 2015; Ishii et al., 2015). The Silkworm - Bombyx 3
mori, (Lepidoptera: Bombycidae) produces a large enough larvae for antifungal drugs 4
distribution studies (Nwibo et al., 2015; Uchida et al., 2016). B. mori larvae were used as 5
a C. tropicalis model of infection in order to evaluate the effective dose of both 6
fluconazole and amphotericin B (Hamamoto et al., 2004). When C. tropicalis was 7
inoculated into the larval hemolymph followed by antifungal drugs administration, it was 8
obtained an effective dose for 50% of them (ED 50%) of 1.8 µg/g of larvae for 9
amphotericin B and fluconazole, being in agreement with previous animal models using 10
mice previously performed (Hamamoto et al., 2004). Therefore, it confirms its possible 11
use for C. tropicalis virulence studies. 12
Drosophila melanogaster larvae (Diptera: Drosophilidae), known as fruit flies also have 13
been used as an animal model to study microbial interactions with innate immune 14
response (Alarco et al., 2004). Zanette and Kontoyiannis satisfactorily used this model to 15
investigate C. tropicalis strains with or without paradoxical growth (Zanette and 16
Kontoyiannis, 2013). 17
Galleria mellonella larvae (Lepidoptera: Pyralidae) have been used as another 18
invertebrate model to investigate fungal and host interactions (Champion et al., 2016), in 19
systemic studies of antimicrobial efficiency (Wei et al., 2016), evaluation of virulence in 20
immunosuppressive models (Torres et al., 2016), immunomodulatory response (Fuchs et 21
al., 2016) and antifungal resistance (Souza et al., 2015). 22
G. mellonella infection by C. tropicalis was used to investigate cross resistance to azoles 23
or multidrug resistance among them and amphotericin B (Forastiero et al., 2013). Two 24
hours after infection with C. tropicalis, different antifungal drugs were applied 25
(fluconazole, voriconazole, amphotericin B and anidulafungin. In this study, 80% of the 26
untreated infected larvae died between day 3 and 4 of infection, while better survival rates 27
were observed for animals inoculated with susceptible strains (10 mg/kg/day of 28
voriconazole and 9 mg/kg/day of fluconazol). When the larvae were infected with strains 29
resistant to the azoles with the same therapeutic doses, survival rates were equivalent to 30
the group that was untreated. This study demonstrates the reliable application of the use 31
53
of the G. mellonella model for the study of infection by Candida as well as for the 1
evaluation of antifungal action (Forastiero et al., 2013). 2
7. Superficial and systemic infections 3
C. tropicalis belongs to the normal human microbiota and is present on the skin, 4
gastrointestinal, genitourinary and respiratory tracts of humans (Basu et al., 2003; Oksuz 5
et al., 2007; Negri et al., 2010). This yeast has been associated with superficial and 6
systemic infections all over the world, specifically in neutropenic patients, or in 7
individuals with a reduction of the microbiota by antimicrobial use or presenting damage 8
in gastrintestinal mucosa (Colombo et al., 2006). 9
C. tropicalis is classified as the third or fourth NCAC species more commonly isolated 10
in the clinical practice (Pfaller et al., 2010; Peman et al., 2012), but it is considered the 11
most prevalent yeast in Asia (Chakrabarti et al., 2009; Kothavade et al., 2010; Adhikary 12
and Joshi, 2011) and the second or the third more isolated species in Brazil and other 13
Latin America countries (20.9% and 13.2%, respectively) (Pfaller et al., 2010). The 14
expressive increase in isolation of this yeast in cases of both superficial and systemic 15
infections in different casuistic all over the world emphasizes its emergent character. 16
The clinical aspects of Candida infections may vary according with the body site affected. 17
Oral candidiasis, VVC and onychomycosis are superficial mycoses caused by this genus, 18
while systemic candidiasis involves blood and deep-seated organs such as the lungs and 19
gastrintestinal tract (Jacobs and Nall, 1990). 20
Oral candidiasis is an opportunistic infection caused by Candida commonly found in the 21
eldery (due to low immunity caused by age), HIV patients, malnourished individuals and 22
those submitted to systemic steroid therapy, denture wearers and people with xerostomia 23
(Muadcheigka and Tantivitayakul, 2015). Clinical manifestations are divided into white 24
and erythematous forms. The white form is characterized by whitish lesions and includes 25
pseudomembranous candidiasis and hyperplastic candidiasis. The erythematous form 26
presents with red lesions, including acute atrophic candidiasis, chronic atrophic 27
candidiasis, median rhomboid glossitis, angular cheilitis and linear gingival erythema. 28
There are also three forms which are not classified into these two clinical categories, 29
which are chronic mucocutaneous candidiasis, cheilocandidiasis and chronic multifocal 30
candidiasis (Millsop and Fazel, 2016). 31
54
In Brazil, a study performed by Silva-Rocha et al. (2014) investigated Candida species 1
distribution of isolates obtained from the oral cavity of kidney transplant recipients from 2
two geographic regions of Brazil (Northeast and South). The authors found that C. 3
tropicalis was the second most prevalent species, corresponding to 4.5% of the isolates. 4
A prevalence study of Candida species obtained from oral candidiasis was carried out in 5
Thailand with 250 strains isolated from 207 patients and C. tropicalis was the third most 6
isolated species (10.4%) (Muadcheigka and Tantivitayakul, 2015). Similarly, in a study 7
conducted in the northwest of Ethiopia with 215 oral cavity isolates from HIV positive 8
patients, this yeast was also the third most prevalent species, with a percentage of isolation 9
equal to 14.1%. More interestingly, 8% of them were resistant to fluconazole and 4% to 10
ketoconazole, itraconazole and fluocytosine (Mulu et al., 2013). Another Indian study 11
concluded that there was a significant increase in Candida infections in oral cancer 12
patients who underwent chemotherapy or radiotherapy, where NCAC species 13
predominated, mainly C. tropicalis, occurring in 42.8% of cases (Jain et al., 2016). 14
VVC is an infection of the vulva and vagina caused by different Candida spp. (Sobel, 15
2016; De Medeiros et al., 2014). C. tropicalis is generally described as the third most 16
prevalent Candida species in VVC, preceded by C. albicans and C. glabrata in most of 17
the studies (Dias et al., 2011; Kanagal et al., 2014; Ragunathan et al., 2014). 18
A study developed in India by Vijaya et al. (2014) with 300 women of reproductive age 19
with clinical signs of VVC reported C. tropicalis as the second most prevalent Candida 20
species, corresponding to 26.4% of the isolates. Of these, 42.9% were resistant to 21
fluconazole and 14.3% to voriconazole. 22
An investigation in Iran with 67 Candida isolates obtained from vaginal secretion samples 23
from patients with VVC found that C. tropicalis was present in 5.9% of cases, with 100% 24
resistance to fluconazole, 50% resistance to clotrimazole, 25% to ketoconazole and 75% 25
against terbinafine. In addition, all isolates showed dose-dependent susceptibility (DDS) 26
to itraconazole (Salahei et al., 2012). 27
Nevertheless, C. tropicalis is reported to a lesser degree in cases of onychomycosis in 28
relation to other species such as C. albicans and C. parapsilosis species complex, 29
promoting paronychial infection mainly in immunosuppressed patients and individuals in 30
extreme age (elderly and children) (Aghamirian et al., 2010; Cambuim et al., 2010). 31
55
A study developed in South Korea reports the prevalence of the Candida genus in 59% 1
of cases of onychomycosis in pediatric patients. The authors obtained 39 isolates, where 2
only 2.6% of them belonged to C. tropicalis (Kim et al., 2013). Another study developed 3
in Mexico analyzing 166 samples of dystrophic nails reports C. tropicalis as one of the 4
less prevalent Candida species in onychomycosis, corresponding to 4.2% of the isolates. 5
However, 14.2% were resistant to fluconazole, itraconazole and ketoconazole (Manzano-6
Gayosso et al., 2011), reinforcing its clinical importance. 7
However, contradictory results were found in a Brazilian study with 200 Candida isolates 8
obtained from nail infections that reported a prevalence of 26% for C. tropicalis. These 9
authors also observed high antifungal drugs resistance in these isolates, including 30.6% 10
resistance to fluconazole, 25% to itraconazole, 9.6% to ketoconazole and 96.2% 11
resistance to terbinafine (Figueiredo et al., 2007). 12
According to McCarty and Pappas (2016), invasive infection by Candida species is 13
commonly associated with medical care, where they may be the third or fourth cause of 14
bloodstream infection (BSI). Risk factors for systemic candidiasis are well known and 15
include the presence of central venous catheter (CVC), the exposure to broad spectrum 16
antibacterial agents, prolonged staying in the ICU with or without mechanic ventilation 17
(more than 3 days), complex surgery, the presence of necrotizing pancreatitis, 18
hemodialysis and immunosuppressive conditions (McCarty and Pappas, 2016). 19
Candidemia caused by C. tropicalis infection has a greater association with skin petechia 20
than other Candida species (Manzano-Gayosso et al., 2011), and this species was 21
described as the most common etiological agent of invasive infection associated with the 22
hospital environment in India (Giri and Kindo, 2012). 23
According to Kontoyiannis et al. (2001), C. tropicalis produces more persistent systemic 24
infections than C. albicans, leading to a longer stay in the hospital environment. Other 25
studies have associated C. tropicalis infections with a higher mortality rate when 26
compared to other NCAC species, even when compared to C. albicans (Kontoyiannis et 27
al., 2001; Kcremery et al., 1999; Eggimann et al., 2003). This factor may be related to 28
the known higher virulence of both species as well as to a higher antifungal resistance by 29
C. tropicalis. 30
56
Recently, an important multicenter study was carried out in 29 Spanish hospitals, where 1
C. tropicalis was isolated in 7.6% of a total of 781 cases of candidemia and 20% of them 2
were resistant to azoles (Guinea et al., 2014). Another multicenter study in China with 3
389 isolates from patients with candidemia admitted to intensive care units found C. 4
tropicalis as the third most isolated species (17.2%), while resistance to fluconazole was 5
observed in 37.3% of isolates of this species, as well as 10% of them were resistant to 6
voriconazole (Liu et al., 2014). 7
A research carried out in Malaysia with 82 bloodstream isolates and peritoneal fluid 8
reports C. tropicalis as responsible for 18.3% of the isolates obtained. Resistance to 9
ketoconazole was observed in 20.9% of the clinical strains, in addition to 13.4% 10
resistance to itraconazole (Santhanam et al., 2013). A study performed by Chang et al., 11
(2013) with isolates from 152 cases of candidemia in Taiwan reported a prevalence of 12
19.7% for C. tropicalis. 13
In Brazil, a study conducted by Oliveira et al. (2010) investigated candidemia in a 14
pediatric hospital in Sao Paulo from 2007 to 2010. C. tropicalis was the second most 15
isolated Candida species (24%), only preceded by C. albicans. More recently, a 16
multicenter surveillance study involving 16 public and private hospitals in the five 17
Brazilian regions (North, Northeast, Center-West, Southeast and South) was conducted, 18
which investigated 137 episodes of systemic infections. NCAC species were responsible 19
for 65.7% of the total infections and C. tropicalis was the third most isolated Candida 20
species (15.3%) (Doi et al., 2016). 21
It is known that candidemia is the most common form of invasive candidiasis, but there 22
are other less frequent clinical manifestations with C. tropicalis as an etiological agent 23
(McCarty and Pappas, 2016). For example, a case of acute disseminated candidiasis in a 24
pediatric patient with aplastic anemia (Fong et al., 1988); the formation of fungal 25
vegetation in a mitral valve prosthesis, causing endocarditis (Nagaraja et al., 2005); and 26
the development of septic arthritis in a cancer patient on chemotherapy with diabetes 27
secondary to corticosteroid therapy that had a negative outcome (Vicari et al., 2003). 28
Another unusual clinical form of candidiasis is endophthalmitis, which is considered an 29
important indicator of systemic infection in hospitalized patients (Donahue et al., 1994). 30
C. tropicalis seems to be an important etiological agent of this infirmity, being classified 31
57
as the fourth species of the genus to promote ocular infection in adult and pediatric 1
patients attended at two medical centers in the USA (Dozier et al., 2011). 2
Disseminated chronic candidiasis is another condition of low occurrence characterized by 3
the presence of histopathological evidence of candidiasis in the spleen, liver and kidneys, 4
or radiological evidence of hepatosplenic or renal candidiasis (Al-Anazi and Al-Jasser, 5
2006). Xu et al. (2010) described the isolation of C. tropicalis in a patient with acute 6
leukemia whose computerized tomography showed multiple hypodense lesions in the 7
liver and spleen. This yeast was also isolated from the kidneys of a patient diagnosed with 8
acute lymphocytic leukemia (Sun et al., 2006) and was associated with a higher mortality 9
rate than other Candida species involved in this disease (Al-Anazi and Al-Jasser, 2006). 10
Finally, C. tropicalis is more rarely found as an etiological agent of respiratory tract 11
infections (Garczewska et al., 2016). This was the second most common yeast species in 12
patients with cystic fibrosis, preceded only by C. albicans. Similarly, another study 13
reports C. tropicalis as the second most common Candida species in cases of pulmonary 14
co-infection with Mycobacterium tuberculosis (Kali et al., 2013). 15
8. Antifungal susceptibility 16
The high incidence of severe infections caused by C. tropicalis has attracted attention, 17
especially considering the evident increase in the reports of resistance of this yeast to 18
antifungal drugs, which is a serious therapeutic problem. 19
Resistance to azoles in this species has been extensively reported, especially to 20
fluconazole. In this respect, Anil and Samaranayake (2003) argue that the increasing 21
global use of this drug is one of the main causes for the dominant tendency of infections 22
caused by NCAC species to the detriment of C. albicans. It is known that there are several 23
factors involved in the development of Candida spp. antifungal resistance in clinical 24
settings, including indiscriminate antifungal therapy use in nosocomial infections 25
(Joseph-Horne and Hollomon, 1997). However, studies on the molecular mechanisms 26
underlying this phenomenon are still necessary. 27
With regard to azoles, the action target of these compounds is the enzyme 14 α-lanosterol 28
demetilase (Erg11p), a product of the ERG11 gene (Table 5), which is part of the 29
ergosterol biosynthesis pathway (Lupetti et al., 2002). Ergosterol is the predominant 30
58
component of the cell membrane of fungi, and influences various cellular functions such 1
as membrane fluidity and integrity, as well as the adequate activity of various enzymes 2
anchored to it, such as proteins related to nutrient transport and chitin synthesis. 3
Therefore, the azoles cause depletion of ergosterol and accumulation of 14 α-methyl 4
steroids harmful to cells, inhibiting growth of fungal cells (Lupetti et al., 2002). 5
Sanglard and Odds (2002) report different mechanisms that may lead to resistance to 6
azoles. The first is the action of multidrug transporters or efflux pumps, which leads to a 7
decrease in drug concentration within the fungal cell (Pfaller, 2012). The positive 8
regulation of MDR1 ("multidrug resistance gene") and CDR1 ("Candida drug resistance") 9
genes (Table 5), are related to the active efflux of azoles in several Candida species, 10
including C. tropicalis (Marie and White, 2009; Morschhauser, 2010).). The induction of 11
efflux caused by CDR genes tends to affect all azoles. In contrast, efflux pumps encoded 12
by MDR genes in Candida are normally selective for fluconazole (Pfaller, 2012). 13
Another pathway leading to azole resistance is the occurrence of amino acid substitutions 14
in Erg11p, which is the target of these drugs, generating changes in protein conformation 15
Forastiero et al., 2013). Increased ERG11 gene expression results in the production of a 16
large amount of 14 α-lanosterol demethylase, favoring the continuous synthesis of 17
ergosterol and the maintenance of cell integrity, which allows the fungus to resist the 18
action of the drugs (Manastir et al., 2011). This factor may occur as a function of a point 19
mutation in ERG11 (Kelly et al., 1993). Pam et al., (2012) detected this point mutation in 20
a C. tropicalis isolate with DDS to fluconazole and demonstrated increased ERG11 21
expression. 22
Eddouzi et al., (2013) studied the molecular mechanisms of drug resistance in a clinical 23
isolate of C. tropicalis with multidrug resistance to fluconazole, voriconazole and 24
amphotericin B, obtained from a hospital in Tunisia. Analysis of sterol production by 25
mass spectrometry and gas chromatography revealed accumulation of 14α-26
methylfecosterol, 4,14α-dimethylzimosterol and 14α-methyl-3β, 6α-diol, indicating 27
change in Erg3p (Table 5). Another study reported the occurrence of ERG3 mutation in 28
a C. tropicalis isolate, with substitution of a phenylalanine for serine in portion 258, a 29
residue that is absolutely conserved in this protein (Vincent et al., 2013). 30
59
A study conducted in five Chinese hospitals investigated resistance to these drugs in 52 1
clinical isolates of C. tropicalis (Jiang et al., 2013). Resistance to fluconazole was 2
observed in 34.6% of the isolates, while 40.4% were resistant to itraconazole and only 3
7.7% to voriconazole. The authors suggest that voriconazole has a more potent activity 4
against the clinical isolates of C. tropicalis than the other drugs tested. 5
Despite the number of studies involving strains of C. tropicalis resistant to azoles, there 6
are still relatively less studies regarding the resistance of this species to other drugs, such 7
as amphotericin B. This compound is the third most commonly used antifungal in clinical 8
practice (Seneviratne et al., 2016) and is part of the class of polyenes. Its fungicidal 9
activity comes from the ability to selectively bind to the ergosterol of the fungal cell, 10
inducing the formation of pores in the plasma membrane, resulting in intense osmotic 11
imbalance and rapid collapse of the cell (Brajtburg et al., 1990). A recent study reported 12
that the production of reactive oxygen species is also part of the fungicidal mechanism of 13
action of amphotericin B (Forastiero et al., 2013). 14
Amphotericin B resistance seems to be a rare phenomenon in yeasts, but Woods and Bard 15
in 1974 demonstrated the development of resistance to this drug in two isolates of C. 16
tropicalis obtained from the urine of a patient with pyelonephritis (Woods and Bard, 17
1974). A subsequent study of these strains revealed the existence of a mutation in the 18
ergosterol of the cell membrane, exactly at the binding site of amphotericin B (Drutz and 19
Lehrer, 1978). Also in the 1970s, Merz and Sandford reported the isolation of eight strains 20
of C. tropicalis resistant to amphotericin B, obtained from urine of transplanted patients, 21
with the same mutation in ergosterol (Merz and Sandford, 1979). 22
Reports of isolation of C. tropicalis resistant to this drug have been progressively 23
increasing over the years. In 1988, Powderly et al., reported that the development of 24
resistance to amphotericin B is most observed in patients with some kind of 25
immunosuppression and who frequently use this drug. Resistance to this polyene is 26
believed to result from changes in ergosterol, or changes in the plasma membrane itself 27
(Seneviratne et al., 2016). 28
Lupetti et al., (2002) postulated that resistance to amphotericin B in Candida species 29
generally occurs due to defects in ergosterol biosynthesis and most likely results from 30
mutations in the ERG3 gene. In addition to the ERG3 gene, mutations in ERG6 can 31
60
generate resistance to polyenes, a phenomenom already described in C. tropicalis 1
(Vandeputte et al., 2007). A study conducted by Forastiero et al., (2013) showed that 2
concomitant mutations in the ERG11 and ERG3 genes lead to multidrug resistance 3
between amphotericin B and azoles (fluconazole and itraconazole) in C. tropicalis. 4
In addition to amphotericin B, echinocandins have been increasingly used in the treatment 5
of invasive infections, being the first new class of echinocandins that target the fungal 6
cell wall, blocking β-1,3-D-glucan synthase (Perlin, 2007). It has been described that 7
these drugs have an excellent range of action against the main Candida species, including 8
C. tropicalis (Pfaller et al, 2008). 9
A surveillance study carried out in 2008 by Pfaller et al., with 5,346 Candida spp. isolates 10
obtained from candidemia infection showed a 12% prevalence for C. tropicalis, and all 11
of them were 100% susceptible for the three echinocandins tested (caspofungin, 12
anidulafungin and micafungin). 13
Despite the extensive use of these drugs for more than a decade, the incidence of 14
resistance in the Candida genus remains very low (Beyda et al., 2012). More recent 15
surveillance studies have indicated an incidence of 2.9 to 3.1% Candida spp. resistance 16
to echinocandins (Castanheora et al., 2010; Arendrup et al., 2010). However, recently 17
Garcia-Effron et al., (2010) reported the isolation of three strains of C. tropicalis resistant 18
to caspofungin obtained from patients with haematological malignancies. A study by 19
Eschenauer et al., (2014) with 185 isolates of C. tropicalis reports 1.4% resistance to 20
caspofungin, anidulafungin and micafungin. 21
Another study described the presence of the paradoxical effect (or paradoxical growth) in 22
15 isolates of C. tropicalis in the presence of high concentrations of echinocandins (Soczo 23
et al., 2007). This phenomenon was first documented by Stevens et al., (2004) for 24
caspofungin in C. albicans, and is defined as fungal growth in the presence of 25
echinocandin concentrations above MIC in broth microdilution susceptibility tests 26
performed according to the guidelines of the Clinical Laboratory Standards Institute 27
(CLSI, previously NCCLS) (Melo et al., 2007). 28
The target enzyme of echinocandins, called glucan synthetase, possesses at least two 29
subunits: Fks1p (encoded by the FKS1, FKS2 and FKS3 genes) and Rho1p (Table 5). 30
Beyda et al., 2012). Fks1p has a catalytic action and Rho1p is a regulatory protein of 31
61
several cellular processes, including the biosynthesis of β-1,3-D-glucan (Chen et al., 1
2011). In general, the reduction of C. tropicalis susceptibility to echinocandins occurs by 2
response to adaptive stress or mutations in the FKS genes. 3
With regard to adaptive responses, it is known that the fungal cell wall is a dynamic 4
structure that presents a compensatory mechanism to increase the production of one or 5
more components that are eventually inhibited, such as that produced by the action of 6
echinocandins. A study by Chen et al., (2014) investigated the role of calcineurin in C. 7
tropicalis, which is one of the main signaling pathways for the compensatory increase of 8
chitin synthesis in C. albicans. Calcineurin is a phosphatase that regulates numerous 9
stress response processes in fungi, including stress promoted on the cell wall (Cowen, 10
2009). The study demonstrated that, in fact, calcineurin is responsible for this effect on 11
C. tropicalis against micafungin, since it promotes the thickening of the chitin layer of 12
the cell wall as a function of β-1,3-D-glucan depletion. 13
In the case of mutations in the FKS1 gene, it is already well established that substitutions 14
in specific gene regions cause reduced susceptibility to echinocandins, being quite 15
associated with therapeutic failure (Perlin, 2007). Mutations in the FKS1 gene in C. 16
tropicalis were already described (Park et al., 2005). Garcia-Effron et al., (2008) 17
demonstrated that 7.5% (3/40) of clinical isolates of C. tropicalis showed resistance to 18
caspofungin because of amino acid substitutions in Fks1p. Jensen et al., (2013) also 19
performed an investigation alterations in FKS1, with isolates of C. tropicalis from patients 20
with acute lymphoblastic leukemia and found that after 8 to 8.5 weeks of treatment with 21
caspofungin, two isolates showed resistance to the three echinocandins. Multilocus 22
sequencing of FKS1 revealed progressive development of heterozygosis, and finally the 23
presence of homozygous mutation, leading to substitutions of amino acids S80P and 24
S80S. 25
The low level of resistance of C. tropicalis to echinocandins and lower side effects, since 26
they target the wall of the fungal cell, make them vital in cases of resistance to fluconazole 27
and amphotericin B, with a broad spectrum of action against C. tropicalis (Pfaller et al., 28
2008). 29
9. Natural products with antifungal properties against Candida tropicalis 30
62
Several groups have been dedicated to the study of products of natural origin with 1
antifungal action, in order to identify and isolate compounds with effective activity, safety 2
in use and low toxicity against pathogenic fungi (Correia et al., 2016). 3
There are several parts of the plants used to search for biological activity, with emphasis 4
on antifungal action, such as leaves (Morais-Braga et al., 2016), stem bark (Mendes de 5
Toledo et al., 2015), roots, seeds and essential oils (Asdadi et al., 2015), that may be 6
isolated used or in synergism with synthetic antifungal drugs, such as fluconazole 7
(Mendes de Toledo et al., 2015). 8
The use of essential oils of Vitex agnus was used in a study by Asdadi et al., (2015) in 9
clinical strains of Candida isolated from hospital infection. The extraction product was 10
tested against Candida isolates using the principle of disc diffusion and broth 11
macrodilution, according to the standardization of CLSI (Salari et al., 2016). It was 12
observed that for the isolates of C. tropicalis, 10 μl of essential oils produced halos of 13
inhibition of growth of 58 mm, superior to the halos of control drugs such as amphotericin 14
B (8 mm), and fluconazole (21 mm) (Salari et al., 2016). 15
Salvia rhytidoa Benth., A plant belonging to the family Lamiaceae, typical in Iran was 16
used to evaluate the antifungal activity in several Candida isolates by Salari et al., (2016). 17
A total of 96 clinical isolates of Candida, including 11 C. tropicalis strains were tested 18
using broth microdilution with the methanolic extract, according to CLSI protocols 19
(Salari et al., 2016). It was observed that for C. tropicalis the MIC range had a variation 20
of 100 - 6.26 μg / mL. Similarly, Siqueira et al., found biological activity using a red 21
propolis alcoholic extract, with an MIC range of 64-32 μg/mL) (Siqueira et al., 2015) for 22
this species. 23
In relation to plants found mainly in Brazilian territory, Correia et al., (2016) carried out 24
an important study with different plants found in the Brazilian Cerrado, a region with an 25
important number of species used in popular medicine, mainly in studies of essential oils 26
with anti-Candida activity (Correia et al., 2016; Nordi et al., 2013). 27
In a study conducted by Morais-Braga et al., (2016) the interaction of aqueous and 28
hydroethanolic extracts of Psidium brownianum was observed in association with 29
fluconazole. The IC 50 values for fluconazole were 68.10 μg/mL for C. tropicalis 30
CTINCQS 40042 and 41.11 μg/mL for C. tropicalis CTLM 23, obtained by broth 31
63
microdilution. When in combination with fluconazole, the aqueous and hydroethanolic 1
extracts of P. brownianum showed a significant reduction in IC 50 values, ranging from 2
37.2-3.10 μg/mL for CTINCM 40042 and 13.66-6.94 μg/mL for CTLM 23. 3
4
All these studies involving the evaluation of vegetal products with biological activity, 5
especially against C tropicalis, has reinforced the great importance and necessity of the 6
emergence of alternative and less toxic sources of treatment, alone or in combinations 7
with different antifungal drugs in commercially available. 8
10. C. tropicalis osmotic stress response and biotechnological applications 9
Several virulence attributes are expressed by fungi in response to stress conditions 10
induced by the environment (Brown et al., 2014), and some yeasts can tolerate high salt 11
concentrations, developing physical and genetic mechanisms to neutralize the two mains 12
deleterious effects of osmotic stress, which are toxicity and loss of water and cellular 13
turgidity (Garcia et al., 1997; Beales, 2004). 14
A study conducted by Rodriguez et al., (1996) reported the gene isolation involved with 15
osmotic adaptation in C. tropicalis, a true homologue of HAL3, called CtHAL3. In fact, 16
C. tropicalis is able to grow in culture medium with more than 10-15% sodium chloride 17
and has been isolated from the hypersaline environment for the first time from Dead Sea 18
samples (Butinar et al., 2005). Bastos et al., (2000) reported the isolation of this yeast 19
from a sample of Amazonian forest enriched with high salt concentration. 20
García et al., (1997) carried out one of the few studies investigating the mechanisms of 21
osmotic adaptation of C. tropicalis, analyzing ion extrusion. The results showed that Na+ 22
/ K+ -ATPase transporters are activated immediately after exposure to hypersaline 23
environment, promoting rapid efflux of ions and restoring intracellular osmotic 24
equilibrium. 25
Exposure to sodium chloride (NaCl) leads to high osmotic stress in fungal cells, 26
promoting rapid loss of water that leads to reduced size and loss of cellular turgidity 27
(Kuhn and Klipp, 2012). 28
With regard to C. tropicalis, García et al., (1997) reported that the accumulation of 29
glycerol necessary for the restoration of a normal cellular physiology occurred only after 30
64
the stationary phase. In addition, they found that there is a preponderant role of efflux 1
pumps in the osmotic adaptation of C. tropicalis to the detriment of the nonionic 2
compensatory mechanisms of water loss and turgor. Besides, the accumulation of 3
intracellular glycerol seems to be less efficient than the activation of the Ion efflux pumps 4
(García et al., 1997). 5
Therefore, C. tropicalis is considered an osmotolerant yeast, since it can grow well in 6
environments with high osmotic pressure, but this condition is not essential to its survival 7
(Tokuoka, 1993). Such property is often associated with its use in industrial and 8
biotechnological practices. 9
In the food industry, osmolytic strains of C. tropicalis improve xylitol production. (Kwon 10
et al., 2006; Misra et al., 2012). Rao et al. (2006) used C. tropicalis strains in hypersaline 11
solution to produce xylitol from corn fiber and sugarcane bagasse. Another example of 12
industrial application of this species is the production of ethanol from algae. (Ra et al., 13
2015) 14
C. tropicalis is still widely used in bioremediation processes. Al-Araji et al. (2007) 15
reported the use of this yeast in the commercial recovery of petroleum spillage. In 2011, 16
Farag and Soliman reported the high degradability of crude oil and hydrocarbons by C. 17
tropicalis. Benard and Tuah (2016) also evaluated this property under conditions 18
simulating sea water. In addition, Yan et al (2005) demonstrated the high potential for 19
degradation of phenol by C. tropicalis in saline medium. Microorganisms with this 20
capacity are called biosorbents, found to correct pollution processes without causing 21
damage to ecosystems (Leitão, 2007). 22
Halotolerance also provides a longer permanence of C. tropicalis in the coastal 23
environment, allowing greater opportunity for contamination of bathers. Prolonged 24
persistence in the marine environment may also lead to adaptation to high concentrations 25
of other ions and UV light. This whole process can be reflected in genetic alterations that 26
results in selection pressure (Krauke and Sychrova, 2008). 27
Recently, our group was involved in the investigation of osmotolerance and its relation 28
to virulence expression in vitro with C. tropicalis isolated from the coastal environment. 29
We found that these strains can fully express virulence attributes and may show a high 30
persistence capacity on the coastal environment, because they all tolerated high salt 31
65
concentration. In addition, they showed high MICs to several antifungal drugs used in 1
current clinical practice, demonstrating that environmental isolates may have pathogenic 2
potential and suggesting that the persistence of yeasts in the sand environment may have 3
leaded to the overexpression of efflux pumps, that may partially explain the reason why 4
C. tropicalis isolates not previously exposed to antifungal drugs had high levels of 5
resistance to azoles and amphotericin B (Zuza-Alves et al., 2016). 6
11. Concluding remarks 7
In conclusion, this review highlights important aspects of C. tropicalis biology and 8
clinical relevance. This species may be easily identified by classical taxonomy, 9
commercial, proteomics and molecular methods and no cryptic sibling species has been 10
discovered. This asexual yeast closely related to C. albicans may be considered of high 11
virulence, which can be verified in animal models of superficial and systemics infections, 12
plus its ability to form true hyphae and complex biofilm in vitro, besides the ability to 13
secret proteinases, phospholipases and hemolisins. C. tropicalis is classified as the third 14
or fourth NCAC species more commonly isolated in the clinical practice, while may be 15
the second more frequently isolated Candida species in Latin America and Asia. Several 16
mechanisms of antifungal resistance have been elucidated, including ERG and FKS gene 17
families’ mutations and efflux pumps. Some natural products have also been investigated 18
as new potential use for future development of antifungal compounds active against C. 19
tropicalis. This species is considered osmotolerant and this characteristic has been 20
recently demonstrated to influence the expression of virulence factors and primary 21
antifungal resistance. This ability to survive to high salt concentrations is a property that 22
explains C. tropicalis potential use for biotechnological processes, including ethanol 23
production through the fermentation of sea algae. Therefore, for all the factors previously 24
described, C. tropicalis may be indubitably considered one of the most important Candida 25
species. 26
Competing interests 27
The authors declare that they have no competing interests 28
Ethics approval and consent to participate 29
Not applicable. 30
66
Consent for publication 1
Not applicable. 2
Availability of supporting data 3
Not applicable. 4
Abbreviations 5
C. albicans: Candida albicans; C. tropicalis: Candida tropicalis; CLSI: Clinical and 6
Laboratory Standards Institute; MALDI-TOF/ MS: Matrix-assisted laser desorption time-7
of-flight mass; ELISA: Enzyme-linked immunosorbent assay. 8
Authors’ contributions 9
DZ and WS prepared the manuscript. GC designed all topics and revised the manuscript. 10
All authors approved the final manuscript. 11
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99
Table 1: Conventional methods used for Candida tropicalis laboratorial identification
Method Principle Advantages Disadvantages References
Cla
ssic
al
met
ho
do
log
y
Auxanogram and
zymogram
Assimilation and fermentation of several different
carbon and nitrogen souces
Easy execution and low cost
Laborious and time-
consuming,
subjectivity of
interpretation
Pincus et al.,
2007;
Sariguzel et al.,
2015
Microculture on cornmeal
agar containing Tween 80
Yeasts incubation on culture medium with Tween
80 and low oxygen tension esporulation and
filamentation
Urease test Urea hydrolysis alkalinizes the medium, causing
the pH indicator to change. The medium goes from
yellow to pink, indicating positivity
Ch
rom
og
enic
med
ia
Chromagar Candida®,
Candida ID2®,
CandiSelect4®, Candida
Brilliance®
Different substrates react with specific enzymes of
the main Candida species and induce the formation
of colonies with different colors for tpresumptive
identification
Rapid screening of different
species and checking the
purity of Candida colonies,
detects mixed infections; high
sensitivity and specificity
Presumptive
identification for only
five species of the
Candida genus
Sariguzel et al.,
2015;
Alfonso et al.,
2010;
Zhao et al., 2016
Sem
i-au
tom
ated
met
ho
ds
API 20C AUX
API ID 32C system
Galleries with different carbon sources, where
growth and assimilation is observed by turbidity in
the respective well
Good reproducibility and easy
execution
May not be completely
accurate on some cases
and may lead to an
incomplete
identification, needing
supplementary tests or
even give a wrong
identification for some
species; higher cost.
Not all the rare
Candida species are
Bowman and
Ahern, 1976;
Stefaniuk et al.,
2016
CandiFast® system The identification well contains cycloheximide,
besides seven carbohydrates, where fermentation is
analyzed after acidification and alteration of media
colors due to the presence of a pH indicator
Used for identification and
antifungal susceptibility
testing
Gundes et al.,
2001
AuxaColor™ Kit Assimilation of 13 sugars, besides the enzymatic
detection of N-acetyl-galactosaminidase,
phenoloxidase and L-proline arilamidase
Good reproducibility and easy
execution
Pincus et al., 2007
Au
tom
a
ted
met
ho
ds Vitek2® System Fluorometric and colorimetric methods for
microorganism’s identification and analysis in a
software which contains a database with 52 yeast
species
Posteraro et al.,
2015
100
BD Phoenix™ Polystyrene strips contain three fluorescent control
wells (a negative and two positives) with 47 wells
containing lyophilized substrates
Rapid results, requires
minimal preparation of
reagentes
included in the
galleries.
Grant et al., 2016
Table 2: Molecular methods and proteomics for the identification and genotyping of Candida tropicalis
Method Principle Advantages Disadvantages References
Pro
teo
mic
s
Protein profile by
mass
spectrophotometry
Uses an ionizing matrix and has been
assembled to automated methods of
microorganisms identification such as
MALDI Biotyper and VITEK-MS and
several other mass spectrometer
Simple methodology of easy
sample preparation and short
time for analysis, more efficient
than the conventional methods,
accurate identification
Higher cost of equipment,
necessity for specialized
training; possible lack of a
robust database.
Sariguzel et al., 2015;
Stefaniuk et al., 2016; Chao
et al., 2014; Keceli et al.,
2016; Angeletti et al., 2015;
Panda et al., 2015
Mo
lecu
lar
iden
tifi
cati
on
Molecular rDNA
sequencing
Based on the ability of DNA polymerase
to copy a DNA strand from the template
in the presence of a primer. The inclusion
of fluorescent markers with different
colors allows the differentiation of the
chains truncated by the respective
fluorescence
Robust technique, automated,
Higher accuracy, gold standard
identification
Requirement for specialized
equipment, expensive
reagents, and highly trained
personnel
Pincus et al., 2007
PNA-FISH Based on the use peptide nucleic acid
probes directed to specific rRNA species
of the main Candida species tagged with
fluorescent dyes
High sensitivity and specificity There may be some problem
in discriminating closely
related microorganisms
Aydemir et al ., 2016;
Stender et al., 2003; Stone
et al., 2013; Calderaro et
al., 2014 Gorton et al.,
2014; Hall et al., 2012
Gen
oty
pin
g
Randomly
Amplified
Polymorphic DNA
(RAPD)
Based on the amplification of DNA
fragments by polymerase chain reaction
(PCR) by using shortprimers containing
random sequences
Fast, simple and low-cost
method for detecting
polymorphisms; Does not
require radioactively labeled
probes; use of arbitrary primers,
no need of initial genetic or
genomic information, and the
Dominant technique; low
reproducibility and low
discriminatory power;
difficult standardization,
possible problems of
interpretation
Almeida et al., 2015
101
requirement of only tiny
quantities of target DNA
Wu et al., 2014
Microsatellites
analysis
Based on the amplification by PCR of
small tandem sequence repeats from 2 to
6 highly polymorphic nucleotides, present
on chromosomal telomeric regions
Easy execution, reproducible,
appropriate for large-scale
epidemiological studies, good
discriminatory power;
Technical challenges during
the construction of enriched
libraries and species-specific
primers
Multilocus
Sequence Typing
(MLST)
Based on the amplification of 6-10
housekeeping genes by PCR, with further
PCR products purification and gene
sequencing. Gene sequencing generates
the sequence type (ST) for haploid
organisms and diploid sequence types
(DST) for the diploids microorganisms,
which also may be compared to a
database
Robust technique with high
discriminatory power, excellent
reproducibility, easy
standardization; data that can be
shared and compared between
different laboratories easily
through the Internet
Requirement for specialized
equipment, expensive
reagents, and highly trained
personnel; phylogenetic
relationships and resolution of
clones can be masked by the
use of slowly evolving
housekeeping genes
Wu et al., 2012; Chen et al.,
2009; Odds and Jacobsen,
2008; Tavanti et al., 2005;
Maiden et al., 1998; Obert
et al., 2007
102
Table 3: Genes recognized as virulence factors in Candida tropicalis. Gene Gene product in
C. tropicalis
Biological function References
Ad
hes
ion
to
epit
hel
ial
cell
s
ALS Als1
Als2
Als3
Adhesin Hoyer et al., 2001;
Punithavathy e
Menon, 2012
HWP1
Hwp1p
Hyphal cell wall adhesin
Wan Harun et al.,
2013
M
orp
hog
enes
is
UME6 Ume6p Positive transcription regulator
responsible for hyphae morphology and
extension; induces HGC1 transcription
Lackey et al., 2013
NRG1 Nrg1p Negative transcription regulator;
inhibiting filamentation
HGC1 Hcg1p Forms a complex between cycline/Cdk
and CDC28 kinase, to inhibit cell
separation and activation of Cdc42
regulator (involved in vesicular
transport in hyphae and actin
polymerization)
Zheng et al., 2007;
Gonzalez-Novo et
al., 2008; Lackey
et al., 2013
PHR1 Phr1p Remodeling of the cell wall, necessary
for maintenance of hyphae shape and
growth, adhesion to abiotic surfaces and
invasion of the epithelium
Calderone et al.,
2010
CDC12 cdc12p septin Formation of the cytoskeleton during
cell growth in filamentation; Binding to
cdc3p actin ligand
Li et al., 2012;
Chang et al., 1997
WOR1 Wor1p Transcription factor that induces
filamentation
Porman et al.,
2013; Slutsky et
al.,1987
Ph
eno
tip
c
swit
chin
g
EFG1 Efg1 Activator or a repressor of hypha
formation
Mancera et al.,
2015
WOR1 Wor1p Master regulator of the white-opaque
switching
Porman et al.,
2013; Slutsky et
al.,1987
Bio
fim
fo
rmat
ion
ALS Als1
Als2
Als3
Adhesin Hoyer et al., 2001;
Punithavathy and
Menon, 2012;
Wan Harum et al.,
2013
HWP1 Hwp1p Hyphal cell wall adhesin
BCR1 Bcr1p Transcription factor for regulation of
adhesin production
RBT5 Rbt5p Filamentation of cells in the biofilm Nobile and
Mitchell, 2006;
Fitzpatrick et al.,
2010
UME6 Ume6p Negative dispersion regulator of biofilm
cells
Uppuluri et al.,
2010
WOR1 Wor1p Negative regulator of mature biofilm
cell release
NRG1 Nrg1p Positive regulator of cells dispersion in
biofilm
ERG11 Erg11p Mechanisms of resistance Lupetti et al., 2012
MDR1 Mdr1p Active drug efflux pump Marie and White,
2009;
103
Morschhauser,
2010 P
rote
inas
e
acti
vit
y SAPT1
SAPT2
SAPT3
SAPT4
Sapt1p
Protein hydrolysis Zaugg et al., 2004;
Silva et al., 2010;
Togni et al., 1996
Ph
osp
ho
lip
ases
acti
vit
y
PLB1
PLC1
Plb1
Plc1
Hydrolysis of ester bonds in glycerol
phospholipids
Hoover et al.,
1998; Bennet et
al., 1998
Hem
oly
tic
acti
vit
y RBT5 Rbt5 GPI-anchored cell-wall protein
involved in hemoglobin utilization
Nobile and
Mitchell, 2006;
Fitzpatrick et al.,
2010
104
Table 4: In vivo models of Candida tropicalis infection
Organism Site of infection References
Mice Lateral tail vein Zhang et al., 2016
Bombyx mori larvae Larval hemolymph Hamamoto et al., 2004; Nwibo et al.,
2015; Uchida et al., 2016
Drosophila melanogaster larvae Injected in the thorax Zanette and Kontoyiannis, 2013
Galleria mellonella larvae Last left proleg Forastiero et al., 2013;
105
Table 5: Genes involved with antifungal resistance mechanisms in Candida
tropicalis Gene Gene product
in C. tropicalis
Biological Function References
Azo
les
ERG11 Erg3p Ergosterol biosynthesis pathway Eddouzi et al., 2013;
Vincent et al., 2013
ERG3 Erg11p Ergosterol biosynthesis pathway Pam et al., 2012;
Manastir et al., 2011;
Kelly et al., 1993
MDR1 Mdr1p Energy-dependent transportation Marie and White, 2009;
Morschhauser, 2010
CDR1 Cdr1p Energy-dependent transportation
Am
ph
ote
rici
n B
ERG3 Erg3p Ergosterol biosynthesis pathway Lupetti et al., 2002;
Forastiero et al., 2013
ERG6 Erg6p Ergosterol biosynthesis pathway Vandeputte et al., 2007
ERG11 Erg11p Ergosterol biosynthesis pathway Forastiero et al., 2013
Ech
ino
cand
ins
FKS1
FKS2
FKS3
Fks1p
Catalytic action
Beyda et al., 2012;
Chen et al., 2011; Park
et al., 2005; Garcia-
Effron et al., 2008;
Jensen et al.,2013
RHO1 Rho1p Regulation of β-1,3-D-glucan
biosynthesis and other cellular processes
106
Fig. 1: Phenotypic characteristics of Candida tropicalis. (A): Brilliant appearance with
slightly fringed border after 48 h of incubation at 30 ° C in Sabouraud dextrose agar; (B):
Colonies with typical dark blue color on CHROMagar Candida® medium after 96 h of
incubation at 35 ° C; (C): Micromorphological aspects after incubation in YPD medium
containing 20% fetal bovine serum (FBS) for 7 days at 30 ° C, 400x: blastoconidia in
single or branched chain, true hyphae and abundant pseudohyphas.
107
Fig. 2: Phylogenetic tree of Candida spp. internal transcribed spacer 1 (ITS1)-5.8S
ribosomal RNA gene and internal transcribed spacer 2 (ITS2) complete sequences and
their accession numbers, obtained from Genbank database at
https://www.ncbi.nlm.nih.gov. Sequences were aligned using BioEdit software (v7.2.61).
Aligned sequences were used for phylogenetic analysis conducted with Mega 7.0.26
Software. The method used for tree constructions was maximum parsimony. Phylogram
stability was accessed by bootstrapping with 1,000 pseudoreplications.
KY673197.1 Candida albicans voucher CA142-W internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and large subunit ribosomal RNA e
KY673196.1 Candida dubliniensis voucher CD129-W internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and large subunit ribosomal e
EF216862.1 Candida tropicalis isolate 16 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene complete sequence and internal transcribed spacer 2 partial sequence
AB109292.1 Candida parapsilosis genes for 18S rRNA ITS1 5.8S rRNA ITS2 28S rRNA partial and complete sequences strain:IFM 52626
KX450873.1 Candida glabrata strain DM 94 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and large subunit ribosomal RNA g...
KY794727.1 Pichia kudriavzevii isolate S16 small subunit ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence e
AF172262.1 Candida lusitaniae internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28S ribosomal RNA gene partial sequence
82
98
64
97
Candida glabrata
Candida dubliniensis
Candida tropicalis
Candida parapsilosis
Candida albicans
Candida krusei
Candida lusitaniae
108
Title: Candida tropicalis geographic population structure maintenance and
dispersion in the coastal environment may be influenced by the climatic season and
anthropogenic action
Diana Luzia Zuza-Alvesa ([email protected]), Walicyranison Plinio Silva-
Rochaa ([email protected]), Elaine C. Franciscob ([email protected]),
Maria Christina Barbosa de Araújoc ([email protected]), Analy Sales de
Azevedo Melob ([email protected]), Guilherme Maranhão Chavesa
aLaboratory of Medical and Molecular Mycology, Department of Clinical and
Toxicological Analyses, Federal University of Rio Grande do Norte, Gal. Gustavo
Cordeiro de Farias street, SN, Petrópolis, Zip code 59012-570, Natal city, RN, Brazil.
bSpecial Micology Laboratory, Department of Medicine, Federal University of São Paulo,
Pedro de Toledo street, 669, 5 floor, Zip code 04039-032, São Paulo city, Brazil.
cDepartment of Oceanography and Limnology, Federal University of Rio Grande do
Norte, Mãe Luiza beach, SN, Via Costeira, Zip code 59014-100, Natal city, 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]
109
Highlights:
MALDI-TOF/MS used as a yeast typing tool shows a relative correspondence with DNA
genotypic typing with microsatellite.
C. tropicalis strains obtained from the coastal environment show high genetic variability,
but population structure may be maintained within the same season.
C. tropicalis dispersion in the coastal environment may occur even at distant geographic
points, probably influenced by anthropogenic action.
Abstract
Candida tropicalis is a pathogenic yeast with worldwide recognition as the second or
third more frequently isolated species in Latin America, for both superficial and systemic
infections. Because of its high prevalence, and growing clinical interest, it is essential to
understand genetic variability patterns of this important Candida species in the tropics.
Besides belonging to the human normal microbiota, C. tropicalis may be found in other
warm blood animals and in the environment, including water and sand of beaches. The
aims of the present study were to evaluate genotypic and phenotypic variability of 62
isolates of C. tropicalis obtained from the coastal environment in Northeast Brazil using
microsatellite and MALDI-TOF/MS comparisons. There was a relatively low
correspondence between these typing techniques employed. Therefore, further studies are
needed to consolidate the use of MALDI-TOF/MS as a yeast typing tool. Nevertheless,
the two methods employed demonstrated the heterogeneity of C. tropicalis in a coastal
environment. We also found relative maintenance of the population structure within the
same season, which may reinforce the idea that this species presents the potential to
remain in the environment for a long period of time. In addition, highly related strains
were found within different geographic points of collection, demonstrating that this strain
may be dispersed at long distances, probably influenced by anthropogenic actions and
driven by the sea tides and wind.
Key-words: Candida tropicalis, genotypic and proteomic typing, coastal environment,
climatic season.
110
1. Short communication
Candida tropicalis is a diploid yeast that has been widely recognized as an emerging
pathogen of great epidemiological importance in Latin America [1] and Asia [2 - 4].
Because of its high prevalence, and growing clinical interest, it is essential to understand
genetic variability patterns of this important Candida species in the tropics [5]. C.
tropicalis belongs to the normal human microbiota and may present on the skin,
gastrointestinal, genitourinary, and respiratory tracts of humans. This yeast has been
associated with both superficial and systemic infections all over the world. C. tropicalis
infection may be acquired from either endogenous or exogenous sources. Candida
tropicalis may also be a commensal yeast of the gastrointestinal tract of birds such as
seagulls and terns, as well as fishes [6]. This yeast has also been isolated from sandy
beaches and coastal waters of The United States and Brazil [7 – 9].
Molecular typing techniques have been employed within strains of the same species for
the investigation of several issues, such as population genetics, reproduction patterns,
sexual recombination, phenotype-genotype relation, infection routes, definition outbreaks
and monitoring of drug resistance [10 – 12] In this sense, microsatellite have been widely
used for the analysis of polymorphisms in fungal species, including C. tropicalis [13, 14].
Microsatellite typing using primer M13, which amplifies short tandem repeats, was first
employed to type Cryptococcus strains in Brazil [15]. In addition, this technique has been
successfully used to type C. albicans strains from several different clinical sources [16].
More recently, Matrix Assisted Laser Desorption/Ionization-Time of flight mass
spectrometry (MALDI-TOF/MS) has been recognized as a rapid, accurate and
economical used for microorganism’s identification. A few studies have also used this
technique as a phenotypic typing method, in a predictive approach for the detection of
resistant phenotypes in bacteria [17] and yeasts belonging to the Candida genus [18, 19].1
ABBREVIATIONS: ERIC-PCR: Enterobacterial Repetitive Intergenic Consensus Sequence -
Polymerase Chain Reaction; MALDI–TOF/ MS: Matrix Assisted Laser Desorption/Ionization-
Time of flight mass spectrometry; MLST: Multilocus Sequence Typing; MSP: Main Spectra
Library; PCR: Polimerase Chain Reaction; RAPD: Randomly Amplified Polymorphic DNA;
UPGMA: Unweighted Pair Group Method using Arithmetic averages
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Recently, our group demonstrated that C. tropicalis strains isolated from the coastal
environment may express important virulence factors in vitro, including adhesion to
epithelial cells, biofilm formation, morphogenesis and lytic enzymes production [9]. In
addition, several strains were markedly tolerant to osmotic stress and resistant to
antifungal drugs used in clinical practice. Here we investigated genotypic and phenotypic
variability of 62 C. tropicalis environmental isolates obtained from different geographical
points of an urban beach located in the northeastern region of Brazil, during two different
climatic seasons (dry season and rainy season). The aim of the present study was to
evaluate the population dynamics of Candida species of medical interest throughout the
year in the coastal environment. It is worth mentioning that we previously described that
most of the strains were able to express virulence factors in vitro and exhibited antifungal
drug resistance [9]. Sand samples were collected in March (dry season; C1; 8 isolates),
April (dry season; C2; 16 isolates) and July (rainy season; C3; 8 isolates), 2012 and in
July (rainy season; C4; 30 isolates), 2013, at six geographically different points of the
beach, selected by the highest concentration of people engaged in recreational activities
(Table 1).
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Table 1: Climate conditions and geographic coordinates of the six sand geographic collection points in Ponta Negra beach, Natal, Rio Grande
do Norte State, Brazil.
CLIMATE CONDITIONS
Dry season Rainy season
Collection 1 Collection 2 Collection 3 Collection 4
Rainfall (mm)1 60.7 146.1 302.1 430.6
Geographic coordinates2 Sand temperature (ºC)3
Point 1 5°53'2.10"S 35° 9'54.05"W - - - 25 ºC
Point 2 5°53'2.00"S 35° 9'60.00"W 36.5 ºC 30 ºC 26 ºC 26 ºC
Point 3 5°52'59.00"S 35°10'8.00"W 37 ºC 30 ºC 29 ºC 26 ºC
Point 4 5°52'34.15"S 35°10'30.88"W 41 ºC 31.5 ºC 29 ºC 27.5 ºC
Point 5 5°52'27.91"S 35°10'34.57"W - - - 28.5 ºC
Point 6 5°52'19.00"S 35°10'38.00"W - - - 29 ºC
1 Data obtained from the National Institute of Meteorology Network
2 Geographic coordinates obtained by GPS (GARMIN eTrex Vista HCx)
3 Temperature calibrated by infrared digital thermometer (MT-360)
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For C. tropicalis identification, yeast colonies were plated on CHROMagar Candida
(CHROMagar Microbiology, Paris, France) to check for purity and screening for different color
colonies. Species identification was based on the characteristics of the cells observed
microscopically after cultivation on corn meal agar containing Tween 80, as well as classical
methodology [20]. Subsequently, the strains were identified with MALDI-TOF/MS technique.
For C. tropicalis genotyping, genomic DNA was extracted using PrepMan® Ultra Protocol
according to the manufacturer’s instructions (Applied Biosystems) [21], Microsatellite typing
was performed using the primer M13 (5’-GAGGGTGGCGGTTCT--3’) (IDT) as previously
described [15]. PCR products were size-separated by agarose gel electrophoresis (1.2 %
agarose) for an initial step of 30 min at 100 V followed by a period of 4.5 h at 55 V. The gel
was stained in a 0.5 µg mL−1 ethidium bromide buffer solution (1 × Tris-Acetate-EDTA). Gel
images were analyzed with the GelCompar II software (Applied Maths). The similarities
between the profiles were calculated using the Dice coefficient. For profile clustering, the
unweighted pair-group method with arithmetic averages (UPGMA) with a tolerance of 2 % was
used [20]. Genetic relatedness analysis classified isolates with indistinguishable fingerprints as
identical; isolates with a genetic similarity coefficient greater than or equal to 90% were
considered highly related and isolates with a similarity coefficient between 80 and 90% were
classified as moderately related. In addition, isolates with a coefficient of similarity between 75
and 80% were grouped within the same cluster, according to criteria established by Soll (2000)
[10].
For MALDI-TOF/MS identification, proteins were extracted with formic acid according to an
adapted protocol [22, 23]. Six hundred microliter of yeast cells in a concentration of 106 were
combined with 7 μL of formic acid 70% in a 1.5 mL micro centrifuge tube. The suspension was
vortexed for 20s and immediately transferred to a reading plate (Bruker Daltonics – USA).
After evaporation, 0.5 μL of a matrix solution (10 mg/mL acid alpha-cyan-4-
Hydroxycinnamicin ethanol: water: acetonitrile [1:1:1]; Sigma – USA) with 0.03%
trifluoroacetic acid were added and gently mixed. The crystallization step occurred at room
temperature and the isolates were analyzed in triplicate. Protein readings were performed with
a Microflex LT mass spectrometer using the FlexControl 3.0 tool (Bruker Daltonics, USA).
Profiling generation was performed using Biotyper 3.0 and Biotyper Real Time Classification
softwares (Bruker Daltonik GmbH). The hierarchical cluster analysis was performed of the
BioTyper 3.0 software package using default correlation function. Basing on the values
obtained from the pairwise comparison of different spectra, a dendrogram was generated
allowing the visualization of similarities among spectra profiles. In the MSP dendrogram,
114
relative distance between isolates is displayed as arbitrary units. Zero indicates complete
similarity and 1.2 indicates high dissimilarity. The arbitrary distance level of 0.8 was chosen
for isolates clustering evaluation.
Dendrogram generated with microsatellite technique yielded 9 different clusters, while
MALDI-TOF/MS showed 6 different clusters (Fig. 1 and Fig. 2). Genetic analysis based on
microsatellite technique revealed a trend of grouping several isolates within the same cluster if
they were collected in the same period (Fig. 1). For instance, Cluster II is composed by all the
strains obtained in the first collection (C1), with the exception of a single isolate (LMMM807).
Cluster V and VIII, almost exclusively grouped isolates obtained from C4 (70% and 83.33%,
respectively). A similar trend was observed in the dendrogram generated with MALDI-
TOF/MS technique, where 81.25% of Cluster II and 83.33% of Cluster VI are composed of
isolates also obtained from C4 (Fig. 2).
115
Fig. 1. Unweighted pair-group method with arithmetic averages dendrogram with 2% of tolerance of 62
strains of Candida tropicalis environmental isolates collected at Ponta Negra beach, Natal, Rio Grande
do Norte State, Brazil from 2012 to 2013. Light grey highlights represent isolates obtained in the first
116
collection. Medium grey highlights represent isolates obtained in the rainy season. Dark grey highlights
represent isolates obtained from geographic point 5.
117
Fig. 2. MSP dendrogram with relative distance between isolates displayed as arbitrary units. Zero
indicates complete similarity and 1.2 indicates high dissimilarity. The arbitrary distance level of 0.8 was
118
chosen for isolates clustering evaluation. Dendrogram represents the main spectra of 62 strains of
Candida tropicalis environmental isolates collected at Ponta Negra beach, Natal, Rio Grande do Norte
State, Brazil from 2012 to 2013. Light grey highlights represent isolates obtained in the first collection.
Medium grey highlights represent isolates obtained in the rainy season. Dark grey highlights represent
isolates obtained from geographic point 5.
If we take the whole picture, the agreement between both techniques is low, because only 27
strains (43.5%) were placed in the same cluster in both microsatellite dendrogram as in
MALDI-TOF/MS dendrograms. On the other hand, interesting findings were observed.
Microsatellite Cluster I is composed by 90% of the strains included in either Cluster 2 or 4
(MALDI-TOF/MS). Microsatellite Cluster II is enriched by MALDI-TOF/MS Cluster III
strains (9 out 13; 38%). Microsatellite Cluster III is composed by 75% of the strains grouped
as Cluster I by MALDI-TOF/MS analysis. Microsatellite Cluster IV and V are enriched with
MALDI-TOF/MS Cluster VI strains (8 out of 13; 62% strains). Microsatellite Clusters VI and
VII are exclusively composed by MALDI-TOF/MS Clusters II and IV (predominantly Cluster
II; 66.6%). Microsatellite Clusters VIII and XIX had either very mixed MALDI-TOF/MS
clusters or not enough strains for comparisons (Figs. 1 and 2).
This finding reflects the nature of the different approaches used, since proteomic typing detects
phenotypic differences within the same species, whereas genotypic approaches detect intrinsic
genetic variability [24]. It is also noteworthy that some isolates (for instance: LMMM806,
LMMM808 and LMMM809) are considered identical by microsatellite technique, but are
placed in other MALDI-TOF/MS clusters (Figs. 1 and 2). This phenomenon is similar to what
Purighalla et al. (2017) describe for Klebsiella pneumoniae isolates, where two isolates were
considered identical by two different DNA-based typing techniques (Enterobacterial Repetitive
Intergenic Consensus Sequence-Polymerase Chain Reaction (ERIC-PCR) and RandomLy
Amplified Polymorphic DNA (RAPD), but showed distinct grouping with the MALDI-
TOF/MS technique, stressing that it is challenging to compare proteomic methods with
genomic-based methods, since a genotype does not necessarily correlate with a phenotype
expressed by a bacterial strain [25]. Similar results were also described to yeasts [19].
When we evaluated separately the influence of the collection period on the grouping of the
isolates within different clusters by the both techniques employed, we can observe that the
strains obtained in C1 (dry season) were almost exclusively placed within Cluster II of the
dendrogram generated by the microsatellite technique (with exception of isolate LMMM807).
When these isolates were evaluated by the MALDI-TOF/MS technique, they are also positioned
within two clusters closely related (III and IV). Although the other isolates are not placed in
119
clusters exclusively formed by strains obtained in each collection period (C2 to C4), they also
stand closely together in each different dendrogram cluster (regardless of the technique
employed), showing a relative degree of relatedness (Figs. 1 and 2).
It is also possible to observe that isolates obtained from the same geographical point are in most
cases considered either identical or highly related to at least another isolate by both techniques.
This fact reflects the clonal mode of reproduction of this species. This phenomenon is very
remarkable in isolates obtained from geographic point 5, where most strains present more than
90% similarity (Cluster V of microsatellite) and were also placed in the same cluster by
MALDI-TOF/MS. On the other hand, the different clusters obtained by both techniques are
composed by isolates obtained from different collection points, demonstrating that dispersion
of highly related strains across several parts of the beach may occur (for instance: strains
LMMM806 and LMMM808, collected from points 2 and 3, respectively). Of note, geographic
collections points are separated from 180 meters to 1 km of distance (Figs. 1 and 2) .
Another interesting finding is that the different isolates are grouped clearly according to the
seasons of the year in which they were isolated, with rare exceptions. Although high degree of
genetic relatedness or protein spectra profiles can occur between strains obtained from the
different seasons of the year, these results are anecdotal, because it only happens to a very few
strains (Figs. 1 and 2).
To our knowledge, this is the first study to show evidence of geographic clustering as a function
of climatic seasonality among environmental isolates of a pathogenic Candida species of
clinical importance, both by genotyping and protein profiling. In fact, the temperature and
rainfall conditions of the two different collection periods were very different (table 1), which
may have promoted the emergence of strains better adapted to each of these climatic conditions.
It is known that C. tropicalis is well adapted to the environmental conditions offered by the
coastal ecosystem [9] and can remain viable in nature for longer than C. albicans [7]. In
addition, the sand of the beach presents filtration properties, because their particles have in their
format cracks and crevices that can function as protected micro-habitats, rich in nutrients,
therefore serving as survival and growth of yeasts [7]. Such a typical characteristic of this
ecosystem can allow the maintenance of a strain in the environment and its subsequent
adaptation to the different temperature conditions, incidence of UV light, rainfall indexes, ionic
concentration, etc. This may lead to the generation of genetic variability within a species, since
the adaptive mechanisms may be reflected in genetic alterations [26]. Therefore, high genetic
120
variability can be attributed not only to microevolutions promoted by prolonged colonization
of microorganisms well adapted to the environment [12].
C. tropicalis has the potential to spread to long distance between geographical regions, probably
through human activity [5]. In the present study, we could realize that strains obtained from
different geographic points (separated by at least 180 m apart) may be highly related. It is
necessary to consider the contribution of the intense anthropogenic action for C. tropicalis
dispersal since this species belongs to the human microbiota and the beach in question is of
great tourist visitation, being frequented by people from various parts of the world, besides
being notoriously polluted (domestic sewage pipes being thrown directly onto the beach), which
can contribute to the population heterogeneity observed in this work.
According to Wu et al. (2014), microsatellite markers are appropriate method for large-scale
studies of the epidemiology of C. tropicalis, with a discriminatory potential similar to
Multilocus Sequence Typing (MLST). In fact, it has been described that different microsatellite
genotypes are related to the continent or country of origin of the isolate [27], as well as isolates
obtained from the same sample or anatomical site, generally present similar genotypes [28]. A
previous study performed by Chaves et al. (2012) with microsatellite genotyping using M13
locus of 51 strains of C. albicans revealed that patients who developed candidemia had highly
related colonizing (obtained from different anatomic sites) and bloodstream isolates, but they
were considered unrelated when different sets of strains were obtained from each patient were
compared, reinforcing the discriminatory power of the technique [16].
The use of MALDI-TOF/MS for epidemiological purposes and typing is still incipient, but
although DNA-based methods are the gold standard for this type of study, this technique offers
a reliable, but faster and more economical way for the identification of important pathogens of
medical interest [29].
Recently, a study conducted by Mlaga et al. (2017) reported the use of proteomic typing to
analyze an outbreak of urinary tract infection by Staphylococcus saprophyticus in southeastern
France, showing a restricted geographic distribution among isolates from two different cities,
which presented distinct clusters [30]. Similarly, Dhieb et al. (2015) reported that Candida
glabrata strains obtained from France and Tunisia were grouped separately with MALDI-
TOF/MS typing. In addition, these authors report that the clusters evidenced with proteomics
technique agreed with the clusters found with microsatellite markers used for the same isolates
[19].
121
Girard et al. (2016) reported the discrimination of Candida auris strains of different geographic
origins within the dendrogram generated by MALDI-TOF/MS, but also reported some
discrepancies. For instance, there are some dendrogram overlaps between Indian and South
African strains, while isolates obtained from Brazil, Korea and Japan form distinct subgroups
[31]. In another study, Prakash et al. (2016) also demonstrated a geographic clustering in
MALDI-TOF/MS typing in nature, although it has not been completely correlated with the
genotyping methods also applied in the work for C. auris [24].
This was also observed in our results for environmental isolates of C. tropicalis, since we found
a relatively low congruence among the typing techniques employed, because only 27 strains
(43.5%) were grouped in the same cluster in both the microsatellite and MALDI-TOF/MS
dendrograms
Finally, we conclude that although there were evident geographic clusters of environmental
isolates of C. tropicalis in both the microsatellite dendrogram and MALDI-TOF/MS, there was
a relatively low correspondence between these typing techniques employed. Therefore, further
studies are needed to consolidate the use of MALDI-TOF/MS as a yeast typing tool.
Nevertheless, the two methods employed demonstrated the heterogeneity of C. tropicalis in a
coastal environment, as well as high genetic variability, probably due to microevolution of the
strains trying to adapt to the environmental conditions. We also found relative maintenance of
the population structure within the same season, which may reinforce the idea that this species
presents the potential to remain in the environment for a long period of time. This fact, together
with our previous publication showing the ability of the strains to express virulence factors
reinforce the pathogenic potential of C. tropicalis in the coastal environment.
DECLARATIONS OF INTEREST: The authors declare no conflicts of interests.
FUNDING: This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.
ACKNOWLEDGEMENTS
122
We are very grateful to Professor Arnaldo Colombo for the donation of Candida spp. reference
strains. We also thank Daniel Kacher, from the Department of Biophysics, Federal University
of São Paulo, for the help with MALDI -TOF analysis.
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6 CONCLUSÕES
O artigo de revisão “An update on Candida tropicalis based on basic and clinical
approaches” destacou aspectos importantes da biologia e relevância clínica de C.
tropicalis:
• C. tropicalis pode ser facilmente identificada por taxonomia clássica,
métodos comerciais, proteômicos e moleculares;
• É uma levedura assexuada intimamente relacionada a C. albicans e
demonstra elevado potencial de virulência;
• É classificada como a terceira ou quarta espécie de CNCA mais comumente
isolada na prática clínica;
• Vários mecanismos de resistência antifúngica em C. tropicalis foram
elucidados, incluindo mutações gênicas e bombas de efluxo;
• Produtos naturais também tem sido estudados para desenvolvimento de
novos medicamentos frente a C. tropicalis.
• C. tropicalis é considerada osmotolerante, propriedade que modula a
expressão de fatores de virulência e resistência antifúngica, além de
proporcionar potencial uso em processos industriais e biotecnológicos;
• C. tropicalis é indubitavelmente uma das espécies mais importantes do
gênero Candida.
Com relação ao artigo “Candida tropicalis geographic population structure
maintenance and dispersion in the coastal environment may be influenced by the
climatic season and anthropogenic action”, concluímos que:
• Embora existam clusters geográficos evidentes de isolados ambientais de C.
tropicalis no dendrograma gerado por microssatélites e no MALDI-TOF/MS,
127
houve uma correspondência relativamente baixa (43,5%) entre essas técnicas
de tipagem empregadas;
• Os dois métodos empregados demonstraram a heterogeneidade de C.
tropicalis em ambiente costeiro, bem como alta variabilidade genética,
provavelmente devido à microevolução de cepas que tentam se adaptar às
condições ambientais;
• Há relativa mantenção da estrutura populacional de C. tropicalis em uma
mesma estação (e mesmo em estações diferentes), o que pode reforçar a ideia
de que essa espécie apresenta potencial de permanecer no ambiente por um
longo período de tempo. Esse fato, aliado à já documentada capacidade das
cepas de expressar fatores de virulência, reforçam o potencial patogênico de
C. tropicalis no ambiente costeiro;
• São necessários mais estudos para consolidar o uso de MALDI-TOF/MS como
ferramenta de tipagem de leveduras, uma vez que essa metodologia
demonstrou baixa concordância quando comparada à genotipagem por
microssatélite, técnica esta já consolidadada para atipagem molecular de
Candida spp.
128
7 COMENTÁRIOS, CRÍTICAS E SUGESTÕES
O trabalho foi desenvolvido como esperado, sem grandes mudanças no projeto
inicial. Graças à valiosa colaboração de alguns professores membros do Programa de
Pós-graduação em Ciências da Saúde da UFRN e outros colaboradores externos,
conseguimos realizar os objetivos propostos para esse estudo, restando ainda outro
artigo que está em fase de preparação para ser submetido à publicação.
Convém destacar a importância do auxílio financeiro recebido para realização
de visita técnica à outras instituições. Além de contribuir diretamente na obtenção de
dados para este estudo, as visitas proporcionaram contato com outras técnicas e
outros pesquisadores, consolidando colaborações e agregando conhecimento
fundamental.
Torna-se importante mencionar que este é o primeiro estudo a mostrar
evidências de agrupamento geográfico em função da sazonalidade climática entre
isolados ambientais de uma espécie de Candida de importância clínica.
129
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