Journal of Medical Microbiology Yeasts from the microbiota of bats: a focus on the identification and antimicrobial susceptibility of cryptic species of Candida --Manuscript Draft-Manuscript Number:
Yeasts from the microbiota of bats: a focus on the identification and antimicrobial susceptibility of cryptic species of Candida
Identification of yeasts from bats
Microbial Ecology and Health
Raimunda S. N. Brilhante Federal University of Ceará Fortaleza, BRAZIL
Raimunda S. N. Brilhante
Order of Authors:
Raimunda S. N. Brilhante José E Maia-Júnior Jonathas S. Oliveira Glaucia M.M. Guedes Aline L. Silva Francisco B.P. Moura Jamille A. Sales Debora S.C.M. Castelo-Branco Jose J.C. Sidrim Rossana A. Cordeiro Waldemiro A. Pereira - Neto Marcos F.G. Rocha
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Manuscript Including References (Word document)
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Journal of Medical Microbiology – Correspondence Article – 2nd Version
Yeasts from the microbiota of bats: a focus on the identification and antimicrobial
susceptibility of cryptic species of Candida
Raimunda S.N. Brilhante1*, José E. Maia-Júnior2, Jonathas S. Oliveira1, Glaucia M;M.
Guedes1, Aline L. Silva2, Francisco B.P. Moura2, Jamille A. Sales2, Débora S.C.M.
Castelo-Branco1, José J.C. Sidrim1, Rossana A. Cordeiro1, Waldemiro A. Pereira-Neto3,
Marcos F.G. Rocha1,2
Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal
University of Ceará, Fortaleza-CE, Brazil.
University of Ceará, Fortaleza-CE, Brazil.
Department of Pathology and Legal Medicine, School of Medicine, Specialized
School of Veterinary Medicine, Postgraduate Program in Veterinary Sciences, State
Department of Transport Engineering, Federal University of Ceará, Fortaleza-CE,
Running title: Identification of yeasts from bats
Keywords: Bats, yeasts, Candida cryptic species, molecular identification, antifungal
*Corresponding author: Raimunda Sâmia Nogueira Brilhante. Rua Barão de Canindé,
210; Montese. CEP: 60.425-540. Fortaleza, CE, Brazil. Tel: +55 85 3366 8319; e-mail:
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Bats harbor several pathogens that can be disseminated through their feces, hence,
becoming important sources of environmental contamination, once they are able to fly long
distances (Botelho et al., 2012). Yeasts colonize the gastrointestinal tract of different animal
species (Brilhante et al., 2013), but reports on the composition and antifungal susceptibility
of the yeast microbiota of bats are scarce. Therefore, this study aimed at isolating yeasts
from bats and their droppings, investigating the occurrence of the cryptic species Candida
albicans-Candida dubliniensis, C. parapsilosis complex, C. famata complex and C.
guilliermondii complex, and, assessing the antifungal susceptibility of the recovered isolates.
This project was approved by Chico Mendes Institute of Biodiversity (license 45268-1)
and the Ethics Committee for the Use of Animals of the State University of Ceará
(protocol 4797909/2014). Animals were captured in Fortaleza and Metropolitan Region,
state of Ceará, Brazil, from January to April 2015, with mist or dip nets. Bat species
were identified according to Reis et al. (2007).
Samples were collected from oral and rectal cavities of the bats with sterile
swabs (Brilhante et al., 2013). Guano samples were collected from shelters, processed
(Filiú et al., 2002) and seeded on Sabouraud agar with chloramphenicol (0.05 g/L),
Sabouraud agar with chloramphenicol (0.05 g/L) and cycloheximide (0.4 g/L), and bird
seed (Guizotia abyssinica) agar. The plates were incubated at 25 °C, for 20 days. Yeast
colonies were identified through growth on chromogenic medium, biochemical and
micromorphological features (Brilhante et al., 2013; Kurtzman et al., 2011). The strains
were also identified through Vitek2™ (Biomerieux). The cryptic species C. albicans/C.
dubliniensis and the species within the complexes C. parapsilosis, C. famata and C.
guilliermondii were, then, identified through molecular methods. DNA was extracted
with High Pure PCR Template Preparation Kit™ (Roche, Germany). C. albicans-C.
dubliniensis were identified through conventional PCR, based on amplicon size, as
described by Ahmad et al. (2012). Restriction fragment length polymorphism (RFLP) 2 Downloaded from www.microbiologyresearch.org by IP: 18.104.22.168 On: Mon, 16 Jan 2017 16:20:06
was used to identify the species within the C. parapsilosis (Tavanti et al., 2005) and C.
famata and C. guilliermondii complexes (Feng et al., 2014). The SADH gene was
amplified, resulting in 716 bp-amplicons that are compatible with C. parapsilosis.
Afterwards, these amplicons were digested with the enzyme BanI (TermoLab –
Lithuania) and the identification of C. orthopsilosis, C. parapsilosis sensu stricto and C.
metapsilosis was based on number and molecular weight of DNA bands (Tavanti et al.,
2005). As for C. famata and C. guilliermondii species complexes, a specific ITS region
was amplified, resulting in 1.1-kb-amplicons that are compatible with C. famata/C.
guiliermondii. These amplicons were digested with the enzymes BsaHI and XbaI
(TermoLab– Lithuania), and the identification of C. famata and Debaryomyces
nepalensis, and C. guilliermondii and C. fermentati was based on number and molecular
weight of DNA bands (Feng et al., 2014). Finally, uncommon Candida isolates were
submitted to sequence analyses of the ITS region (Desnos-Ollivier et al., 2008).
The minimum inhibitory concentration (MIC) of antifungal drugs against 32 of
the isolated yeasts was determined through broth microdilution method (CLSI, 2008a).
Amphotericin B, itraconazole, voriconazole and caspofungin were tested. MIC values
were interpreted as recommended (CLSI, 2008b; CLSI, 2012). C. parapsilosis ATCC
22019 and C. krusei ATCC 6258 were included as quality control.
Pearson's chi-square test, Fisher's exact test and Kruskal–Wallis one-way
ANOVA, followed by Dunn's post-hoc test, were applied in the analyses of the obtained
data. P-values lower than 5% indicated significant conclusions.
Fifty-six bats (3 Artibeus lituratus; 39 Carollia perspicillata; 1 Diphylla
ecaudata; 6 Glossophaga soricina; 2 Phyllostomus stenops; 1 Peropteryx macrotis; 1
Saccopteryx bilineata; 3 Trachops cirrhosus) were captured and 26 (46.4%) were
positive for the recovery of yeasts from at least one site. Yeasts were not recovered from 3 Downloaded from www.microbiologyresearch.org by IP: 22.214.171.124 On: Mon, 16 Jan 2017 16:20:06
the bat species Diphylla ecaudata and Phyllostomus stenops. A total of 34 isolates were
obtained: 26 from the rectum and 08 from the oral cavity. Furthermore, four (33.3%) of
the 12 guano samples were positive, with one yeast species per specimen (Table 1). The
rectum had a higher recovery rate than the oral cavity (P