Journal of Medical Microbiology

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Journal of Medical Microbiology. Yeasts from the microbiota of bats: a focus on the identification and antimicrobial susceptibility of cryptic species of Candida.

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:

JMM-D-16-00378R1

Full Title:

Yeasts from the microbiota of bats: a focus on the identification and antimicrobial susceptibility of cryptic species of Candida

Short Title:

Identification of yeasts from bats

Article Type:

Correspondence

Section/Category:

Microbial Ecology and Health

Corresponding Author:

Raimunda S. N. Brilhante Federal University of Ceará Fortaleza, BRAZIL

First Author:

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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Click here to download Manuscript Including References (Word document) Brilhante et al. 2016 - 2nd Version without

Journal of Medical Microbiology – Correspondence Article – 2nd Version

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Yeasts from the microbiota of bats: a focus on the identification and antimicrobial

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susceptibility of cryptic species of Candida

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Raimunda S.N. Brilhante1*, José E. Maia-Júnior2, Jonathas S. Oliveira1, Glaucia M;M.

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Guedes1, Aline L. Silva2, Francisco B.P. Moura2, Jamille A. Sales2, Débora S.C.M.

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Castelo-Branco1, José J.C. Sidrim1, Rossana A. Cordeiro1, Waldemiro A. Pereira-Neto3,

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Marcos F.G. Rocha1,2

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Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal

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University of Ceará, Fortaleza-CE, Brazil.

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University of Ceará, Fortaleza-CE, Brazil.

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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,

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Running title: Identification of yeasts from bats

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Keywords: Bats, yeasts, Candida cryptic species, molecular identification, antifungal

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susceptibility

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*Corresponding author: Raimunda Sâmia Nogueira Brilhante. Rua Barão de Canindé,

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210; Montese. CEP: 60.425-540. Fortaleza, CE, Brazil. Tel: +55 85 3366 8319; e-mail:

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[email protected]

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Bats harbor several pathogens that can be disseminated through their feces, hence,

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becoming important sources of environmental contamination, once they are able to fly long

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distances (Botelho et al., 2012). Yeasts colonize the gastrointestinal tract of different animal

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species (Brilhante et al., 2013), but reports on the composition and antifungal susceptibility

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of the yeast microbiota of bats are scarce. Therefore, this study aimed at isolating yeasts

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from bats and their droppings, investigating the occurrence of the cryptic species Candida

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albicans-Candida dubliniensis, C. parapsilosis complex, C. famata complex and C.

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guilliermondii complex, and, assessing the antifungal susceptibility of the recovered isolates.

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This project was approved by Chico Mendes Institute of Biodiversity (license 45268-1)

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and the Ethics Committee for the Use of Animals of the State University of Ceará

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(protocol 4797909/2014). Animals were captured in Fortaleza and Metropolitan Region,

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state of Ceará, Brazil, from January to April 2015, with mist or dip nets. Bat species

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were identified according to Reis et al. (2007).

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Samples were collected from oral and rectal cavities of the bats with sterile

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swabs (Brilhante et al., 2013). Guano samples were collected from shelters, processed

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(Filiú et al., 2002) and seeded on Sabouraud agar with chloramphenicol (0.05 g/L),

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Sabouraud agar with chloramphenicol (0.05 g/L) and cycloheximide (0.4 g/L), and bird

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seed (Guizotia abyssinica) agar. The plates were incubated at 25 °C, for 20 days. Yeast

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colonies were identified through growth on chromogenic medium, biochemical and

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micromorphological features (Brilhante et al., 2013; Kurtzman et al., 2011). The strains

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were also identified through Vitek2™ (Biomerieux). The cryptic species C. albicans/C.

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dubliniensis and the species within the complexes C. parapsilosis, C. famata and C.

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guilliermondii were, then, identified through molecular methods. DNA was extracted

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with High Pure PCR Template Preparation Kit™ (Roche, Germany). C. albicans-C.

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dubliniensis were identified through conventional PCR, based on amplicon size, as

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described by Ahmad et al. (2012). Restriction fragment length polymorphism (RFLP) 2 Downloaded from www.microbiologyresearch.org by IP: 184.174.98.135 On: Mon, 16 Jan 2017 16:20:06

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was used to identify the species within the C. parapsilosis (Tavanti et al., 2005) and C.

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famata and C. guilliermondii complexes (Feng et al., 2014). The SADH gene was

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amplified, resulting in 716 bp-amplicons that are compatible with C. parapsilosis.

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Afterwards, these amplicons were digested with the enzyme BanI (TermoLab –

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Lithuania) and the identification of C. orthopsilosis, C. parapsilosis sensu stricto and C.

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metapsilosis was based on number and molecular weight of DNA bands (Tavanti et al.,

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2005). As for C. famata and C. guilliermondii species complexes, a specific ITS region

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was amplified, resulting in 1.1-kb-amplicons that are compatible with C. famata/C.

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guiliermondii. These amplicons were digested with the enzymes BsaHI and XbaI

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(TermoLab– Lithuania), and the identification of C. famata and Debaryomyces

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nepalensis, and C. guilliermondii and C. fermentati was based on number and molecular

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weight of DNA bands (Feng et al., 2014). Finally, uncommon Candida isolates were

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submitted to sequence analyses of the ITS region (Desnos-Ollivier et al., 2008).

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The minimum inhibitory concentration (MIC) of antifungal drugs against 32 of

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the isolated yeasts was determined through broth microdilution method (CLSI, 2008a).

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Amphotericin B, itraconazole, voriconazole and caspofungin were tested. MIC values

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were interpreted as recommended (CLSI, 2008b; CLSI, 2012). C. parapsilosis ATCC

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22019 and C. krusei ATCC 6258 were included as quality control.

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Pearson's chi-square test, Fisher's exact test and Kruskal–Wallis one-way

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ANOVA, followed by Dunn's post-hoc test, were applied in the analyses of the obtained

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data. P-values lower than 5% indicated significant conclusions.

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Fifty-six bats (3 Artibeus lituratus; 39 Carollia perspicillata; 1 Diphylla

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ecaudata; 6 Glossophaga soricina; 2 Phyllostomus stenops; 1 Peropteryx macrotis; 1

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Saccopteryx bilineata; 3 Trachops cirrhosus) were captured and 26 (46.4%) were

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positive for the recovery of yeasts from at least one site. Yeasts were not recovered from 3 Downloaded from www.microbiologyresearch.org by IP: 184.174.98.135 On: Mon, 16 Jan 2017 16:20:06

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the bat species Diphylla ecaudata and Phyllostomus stenops. A total of 34 isolates were

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obtained: 26 from the rectum and 08 from the oral cavity. Furthermore, four (33.3%) of

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the 12 guano samples were positive, with one yeast species per specimen (Table 1). The

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rectum had a higher recovery rate than the oral cavity (P