Journal of Medical Microbiology Papers in Press. Published March 14, 2013 as doi:10.1099/jmm.0.055566-0 1
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Journal of Medical Microbiology - Original Research - 2nd Version
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Detection of Candida spp. resistant to azoles in the microbiota of rheas (Rhea
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americana): Possible implications for human and animal health
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Raimunda Sâmia Nogueira Brilhante1, Lucas Pereira de Alencar1,2, Rossana de Aguiar
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Cordeiro1, Débora de Souza Collares Maia Castelo-Branco1, Carlos Eduardo Cordeiro
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Teixeira1, Ramila de Brito Macedo1, Daniel Teixeira Lima1, Manoel de Araújo Neto Paiva1,2,
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André Jalles Monteiro3, Nilza Dutra Alves4, Moacir Franco de Oliveira4, José Júlio Costa
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Sidrim1, Marcos Fábio Gadelha Rocha1,2
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Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of
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Ceará, Fortaleza-CE, Brazil.
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Ceará, Fortaleza-CE, Brazil.
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CE, Brazil
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Brazil,
Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical
School of Veterinary, Postgraduate Program in Veterinary Science, State University of
Department of Statistics and Applied Mathematics, Federal University of Ceará, Fortaleza-
Department of Animal Science, Federal Rural University of the Semiarid, Mossoró-RN,
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Corresponding Author. R. S. N. Brilhante. Rua Barão de Canindé, 210; Montese. CEP:
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60.425-540. Fortaleza, CE, Brazil. Fax: 55 85 3295-1736 E-mail:
[email protected]
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Abstract
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There is growing interest in breeding rheas (Rhea americana) in Brazil, however, there are no
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data on the yeast microbiota of the gastrointestinal tract of this avian species and the
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phenotypical characteristics of these yeasts are not known. Therefore, the aim of this work
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was to isolate Candida spp. from the digestive tract of rheas and to evaluate the in vitro
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antifungal susceptibility and secretion of phospholipases of the recovered isolates. For this
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purpose, 58 rheas from breeding operations in the cities of Fortaleza and Mossoró,
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Northeastern Brazil, were used. Samples were gathered from the oropharynx and cloaca of the
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animals, using sterile swabs. Stool samples were collected from their pens, by scraping with a
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scalpel blade. For the primary isolation, the material was seeded onto 2% Sabouraud dextrose
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agar supplemented with chloramphenicol (0.5 g/L). The isolates were identified based on
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morphological and biochemical features. After identification, all the strains were submitted to
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antifungal susceptibility testing, according to the methodology defined by the Clinical
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Laboratory Standards Institute (document M27-A3), against amphotericin B, itraconazole and
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fluconazole. The phospholipase activity the Candida spp. isolates was also tested, by
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culturing on egg yolk agar. Candida spp. were isolated from at least one anatomical site in
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36/58 birds (14/17 juveniles and 22/41 adults) and in 6/10 fecal samples. Mostly, only a
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single species was isolated from each collection site (36/56 positive sites), with up to three
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species being observed only in four cases (4/56). A total of 77 isolates were obtained,
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belonging to the species C. parapsilosis sensu lato (19/77), C. albicans (18/77), C. tropicalis
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(13/77), C. guilliermondii (12/77), C. krusei (10/77) and C. famata (5/77). C. albicans was
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more prevalent in the oropharynx of the juvenile rheas, when compared to adult ones
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(P 12 months). The birds were physically restrained
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and before specimen collection, they were individually evaluated. This study was approved by
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the animal research ethics committee of Ceará State University (protocol number 11044670-
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4/43).
′
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Sample collection. Samples were collected from two anatomical sites (oropharynx and
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cloaca) and from droppings. Sterile cotton swabs were used to obtain samples from the
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oropharynx and cloaca. The swabs were inserted into the anatomical site and rotated. Then
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they were placed into sterile glass slants, containing 1 mL of sterile saline (0.9% NaCl),
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keeping the cotton extremity in contact with the solution at 10 ºC until processing, within 12
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hours. A total of ten stool samples com posed of a pool of feces was collected from the pens
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where the birds were maintained and were kept at 10 ºC in sterile saline solution until
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processing, within 12 hours. The samples were kept refrigerated at 10 °C until laboratory
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processing (Brilhante et al., 2010).
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Isolation and fungal identification procedures. The swabs with samples from the
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oropharynx and cloaca were inoculated in Petri dishes containing 2% Sabouraud dextrose
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agar (SDA, Difco Laboratories) with chloramphenicol and incubated at 25 ºC, for 10 days,
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during which the dishes were observed daily for evaluation of the presence of fungal colonies.
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Previous to streaking agar plates, the stool samples were processed as described by Brilhante
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et al. (2010). The cultured media were incubated at 25 ºC for 10 days. For each positive
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sample, colonies were observed microscopically after Gram staining. For Candida spp., the
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colonies were initially grown on chromogenic media (HiChrome Candida Differential Agar,
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HiMedia, Mumbai, India) for identification of mixed colonies. Afterwards, individual
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colonies were subcultured into slants containing potato dextrose agar and Christensen’s urea
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agar and after 24 to 48h the microorganisms were grown on cornmeal Tween-80 agar for
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morphological analysis. A sugar assimilation test was also performed for each isolate and the
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results were interpreted according to De Hoog et al. (2000).
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Antifungal susceptibility testing. The Candida spp. strains were tested against amphotericin
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B (AMB, Sigma Chemical Corporation, USA), fluconazole (FLC, Pfizer, Brazil) and
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itraconazole (ITC, Janssen Pharmaceutica, Belgium). The antifungal drugs were diluted
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according to the document M27A3 (CLSI, 2008). The concentration range tested was
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0.03125-16 µg/mL for AMB and ITC and 0.125-64 µg/mL for FLC (Brito et al., 2009; Sidrim
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et al., 2010). Inocula were prepared from two-day-old cultures grown on potato dextrose agar
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at 35 oC to reach a final concentration of 0.5 - 2.5 x 103 cells/mL in RPMI medium buffered
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to pH 7 with 0.165M morpholinepropanesulfonic acid (MOPS), as recommended by CLSI
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(2008). Susceptibility was investigated by broth microdilution in 96-well microdilution plates
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at 35 ºC for 48 hours. All the isolates were tested in duplicate. For AMB, MIC was defined as
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the lowest concentration at which no growth was observed; for ITC and FLC, MICs were
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defined as the lowest drug concentration inhibiting fungal growth by 50% when compared to
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the control well (CLSI, 2008). Isolates with MICs >1, 64 and 1 µg/mL were considered ≥
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resistant to AMB, FLC and ITC, respectively (CLSI, 2008), except for C. albicans, C.
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parapsilosis sensu lato and C. tropicalis against FLC, which were considered resistant for
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MIC
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each test (CLSI, 2008).
≥
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Phospholipase experiments. All strains were tested for phospholipase activity. The test was
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performed according to Sidrim et al. (2010). Briefly, the medium used was 2% Sabouraud
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dextrose agar, supplemented with 1 mol/L of sodium chloride, 0.05 mol/L of calcium chloride
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and 8% sterile egg yolk emulsion, at a concentration of 30%. The emulsion was heated to 40
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ºC and incorporated into the sterile Sabouraud medium after it reached the same temperature.
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Then the medium was poured into 90-mm Petri dishes and the yeast inoculum was prepared
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in sterile saline at a concentration of 4 on the McFarland scale. The inoculum (5 µL) was
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dripped onto a 5-mm sterilized filter paper disk, which was then placed on the agar. Next,
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plates were incubated at 35 ºC for seven days. Phospholipase activity (Pz) was determined by
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calculating the ratio between the diameter of the yeast colony and the total diameter, including
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the colony and the precipitation zone. According to this criterion, when Pz = 1, the isolate is
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phospholipase negative; when 1 > Pz
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and when Pz < 0.64, the isolate is strongly positive for this enzyme.
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0.64 the isolate is positive for phospholipase activity;
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Statistical Analysis. Fisher’s exact test was used to check for differences in the number of
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isolates between the anatomical sites and bird age ranges (adult versus juvenile). Student’s t-
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test was used to compare the MICs in relation to the antifungal drugs as well as for
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comparison between the sites in relation to the species isolated for paired samples. To
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compare the species irrespective of collection site, ANOVA was used with post hoc
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application of the Fisher test. ANOVA was also employed to assess differences in production
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of phospholipases, both among species and between their isolation sites. Results with p< 0.05
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were considered significant.
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RESULTS
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A total of 36/58 rheas were positive for growth of Candida spp. in at least one of the
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sites sampled (14/17 for juveniles and 22/41 for adult birds). The positivity per site was 29/58
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in the oropharynx and 21/58 in the cloaca. In 14/58 animals, Candida spp. was recovered
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from both anatomical sites. As for the stool samples, 6/10 were positive.
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Only one Candida species was isolated from 39/56 positive samples, while in 13/56
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cases two species were isolated and in 4/56 three species were isolated (Table 1). Therefore, a
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total of 77 Candida spp. were obtained, comprising six species, as described in Table 2. The
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most frequently isolated species were C. parapsilosis sensu lato (19/77) and C. albicans
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(18/77). C. albicans was more frequent in the oropharynx of juvenile birds than in adults (P
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Pz
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activity average of the strains that were positive was 0.75, with 9/18 C. albicans, 1/5 C.
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famata, 2/12 C. guilliermondii, 4/19 C. parapsilosis sensu lato and 2/13 C. tropicalis. The
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average of Pz in strongly positive strains was 0.60 for 1/12 C. guilliermondii, 3/19 C.
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parapsilosis sensu lato and 1/13 C. tropicalis). No significant difference was found
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concerning the secretion of phospholipases by Candida species.
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0.64) and five were strongly positive (Pz 16 AMB 0.03125 - 0.5 C. famata FLC 0.25 -16 (5) ITC 0.0625 - 0.5 AMB 0.03125 - 0.5 C. guilliermondii FLC 0.5-32 (12) ITC 0.03125 - >16 AMB 0.0625 - 1 C. krusei FLC 0.125 - >64 (10) ITC 0.0625 - >16 C. parapsilosis AMB 0.03125 - 0.5 sensu lato FLC 0.125 - >64 (19) ITC 0.03125 - >16 AMB 0.03125-1 C. tropicalis FLC 0.125 - 32 (13) ITC 0.0625 - >16 AMB: Amphotericin B, FLC: Fluconazole, ITC:
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sensible, R: resistant, (-): not detected.* Not calculated because of the small number of
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strains, which does not allow reliable results.
C. albicans (18)
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0.25 0.25 0.55 18 --33.26 64.00 128.0 3 15 5.879 16.00 32.00 5 13 0.125 * * 5 --3.48 * * 5 --0.143 * * 5 --0.118 0.187 0.425 12 --3.175 4.0 27.20 12 --0.166 0.125 22.55 11 1 0.25 0.25 0.95 10 --4.287 2.00 121.6 8 2 0.267 0.187 28.85 9 1 0.12 0.125 0.25 19 --2.489 4.0 32.0 12 7 0.268 0.125 32.0 16 --0.146 0.125 0.8 13 --3.232 4.0 32.0 7 6 1.0 0.25 32.00 8 5 Itraconazole, GM: Geometric mean, S:
Table 4: Phospholipase activity of Candida spp. strains
Species
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C. albicans C. famata C. guilliermondii C. krusei C. parapsilosis sensu lato C. tropicalis Total
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Phospholipase activity [mean±SD (n)] Positive Strongly isolates positive isolates (1>Pz≥0,64) (Pz < 0,64) 0.74±0.09 (9) --0.65 (1) --0.77±0.14 (2) 0.55 (1) ----0.74±0.06 (4) 0.62±0.01 (3) 0.85±0.07 (2) 0.61 (1) 0.75±0.09 (18) 0.60±0.03 (5)
Negative isolates (Pz=1) 9 4 9 10 12 10 54
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