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Matrobrathium amazonitum: an alternative for microbiological monitoring of aquatic environments in Brazil/Matrobrathium amazonitum uma alternativa para o monitoramento microbiologico de ambientes aquaticos no Brasil.

INTRODUCTION

Several studies have identified Candida spp. as potential biological indicators of environmental degradation (MEDEIROS et al., 2008; BRILHANTE et al., 2011, 2012; CASTELO-BRANCO et al., 2013), particularly in samples obtained from aquatic sources (BUTINAR et al., 2005; MEDEIROS et al., 2008). In these studies, the isolation of this genus was greater than that of other microorganisms, including bacteria, demonstrating the potential use of this yeast for environmental monitoring.

Monitoring aquatic environments requires an adequate water sampling technique, including the selection of representative collection sites and considering environmental factors, such as seasonality, temperature, the water column and the presence of affluent or effluent waters (APHA/AWWA/ WEF, 1998). These requirements may represent an obstacle for the adequate monitoring of fresh water environments, because of the large number of samples needed. Hence, it is important to seek alternatives to facilitate monitoring of aquatic ecosystems. In this context, the use of aquatic crustaceans has been reported as a reliable alternative for that purpose, especially because of their feeding habits (filter feeding) and benthic behavior, as described by VIRGA et al. (2007) and BRILHANTE et al. (2011).

More recently, BRILHANTE et al. (2011) performed a research with the freshwater prawn M. amazonicum (Amazon River prawn) in captivity and from the natural environment for the isolation of yeasts and Candida was the most isolated genus, showing that it belongs to the gastrointestinal microbiota of these animals. In addition, these authors suggested that these prawns may be an important environmental sentinels if they harbor in their gastrointestinal tract Candida spp. from the aquatic environment. Thus, in light of these findings and considering the wide distribution of the species M. amazonicum in South America, the objective of the present study was to evaluate the role of these prawns as carriers of Candida spp. from the aquatic environment.

MATERIALS AND METHODS

Microorganisms

In this study, 27 strains of Candida spp. were analyzed, out of which seven were recovered from wild-harvested M. amazonicum, while 20 were recovered from the aquatic environment and were deposited in our culture collection. It is important to emphasize that the analyzed Candida strains, from animal and environmental sources, were simultaneously recovered.

Of the 20 environmental strains, 13 were isolated from surface water (SW) and seven from sediment (S). These strains belong to the culture collection of the Specialized Medical Mycology Center of the Federal University of Ceara, where they are kept on 2% Sabouraud dextrose agar. They were manipulated under level II biosafety procedures.

Candida spp. from the aquatic environment were obtained from Catu Lake, which is located at the municipality of Aquiraz, Ceara state, Brazil, about 35 kilometers from the state capital. It is delimited by the UTM coordinates 0567000E, 9561273N and 0575000E, 9569000N. It is a rich freshwater body with mangrove areas that have been degraded by uncontrolled occupation of the surrounding area and pollution with residues from industrial, commercial and farming activities (BRILHANTE et al., 2011).

Water samples were collected, according to MEDEIROS et al. (2008). Then, four collection sites were included, as follows: recreational area and prawn collection site (point 1, 3[degrees]55'59.79" S and 38[degrees]21'50.10" W), agricultural wastewater (point 2, 3[degrees]55'47.25" S and 38[degrees]22'14.16" W), industrial wastewater (point 3, 3[degrees]56'03.70" S and 38[degrees]22'25.15" W) and residential area (point 4, 3[degrees]56'56.72" S and 38[degrees]22'31.57" W). Two water samples (SW and S) were monthly collected from each collection site, during one year (from March 2011 to February 2012). Overall, a total of 96 water samples were obtained for this study.

Adults of M. amazonicum were monthly harvested from Catu Lake (point 1) in the same period as the water samples. Afterwards, the digestive tracts of 10 individuals were removed, placed in sterile slants containing sterile saline and treated as one single sample (BRILHANTE et al., 2011). Overall, 12 collections were performed. This study was previously approved by the Chico Mendes Institute for Conservation of Biodiversity/Biodiversity Authorization and Information System--SISBIO, under the number 28175-1.

Microbiological processing

Samples were processed in a biosafety level II cabinet and were seeded on 2% Sabouraud agar plus chloramphenicol (0.5g [L.sup.-1]), in Petri dishes for primary isolation. Water samples were processed according to MEDEIROS et al. (2008) with some modifications. Briefly, a 100-[micro]L aliquot of the SW samples was spread on the medium, after homogenization, while the S samples were processed, after centrifuging for 20 minutes at 3,000rpm. Then, the supernatant was removed and the substrate was suspended again in 2mL of a sterile 0.9% solution of NaCl. Afterwards, the suspension was agitated in a vortex mixer for 3 minutes and left to rest for 30 minutes at 25[degrees]C. Next, 100-[micro]L aliquots of the supernatant of each sample were seeded on the culture medium. The digestive tracts of adult prawns were processed as described by BRILHANTE et al. (2011) and seeded onto the culture medium. The inoculated Petri dishes containing the culture media were incubated at 25oC, for 10 days, and were daily observed (BRILHANTE et al., 2011).

The yeast colonies were identified through specific morphological and biochemical tests, including growth on chromogenic medium (CHROMagar Candida, BD, USA), micromorphology on cornmeal-Tween 80 agar, carbohydrate and nitrogen assimilation and urease production (BRILHANTE et al., 2011), and the results were interpreted according to (DE HOOG et al., 2000). Strains that presented dubious identity were also identified through VITEK II automated system (BioMerieux, USA). Additionally, the susceptibility of these microorganisms to amphotericin B, fluconazole and itraconazole was evaluated through broth microdilution method. Minimum inhibitory concentrations (MIC) of >1, [greater than or equal to] 64, [greater than or equal to] 1[micro]g [mL.sup.-1] were considered resistant to amphotericin B, fluconazole and itraconazole, respectively (CLSI, 2008).

M13-PCR fingerprinting and OPQ-16 RAPD

The DNA from the strains was extracted after 48 hours of growth on potato dextrose agar, according to the methodology described by CASTELO-BRANCO et al. (2013).

For molecular comparison between the Candida isolates from the aquatic environment (SW and S) and from prawns, the PCR-fingerprinting technique was used, according to the method described by CASTELO-BRANCO et al. (2013), using the single primer M13 (59-GAGGGTGGCG GTTCT-39) and the PCR mix (25 [micro]L), containing 10mM of Tris/ HCI (pH 8.3), 50mM of KCl, 1.5mM of MgCl2, 0.2mM of dNTPs, 0.15mM of the primer, 2.5U of Taq polymerase (MBI Fermentas) and 25ng of yeast DNA. The RAPD reactions were performed with the primer OPQ16 (5' AAGAGCCCGT3'), according to the method described by CASTELO-BRANCO et al. (2013). The RAPD reaction was carried out with a total volume of 10[micro]L, containing 50ng of genomic DNA, 1X buffer, 1mM of Mg[Cl.sub.2], 2pmol of primer, 0.5mM each of deoxynucleoside triphosphate and 1 U [micro][L.sup.-1] of Hot Start Taq polymerase.

Dice similarity coefficient was measured and a dendrogram was obtained through the use of the Unweighted Pair Group Method with Arithmetic Average (UPGMA), through the software BioNumerics (version 6.6), resulting in the analysis of clusters and measure of relatedness among isolates.

RESULTS

Data referring to the identity, the origin and the antifungal susceptibility profile of the recovered Candida strains are listed in table 1. Five to eight DNA bands were generated through the M13-PCR fingerprinting, while three to ten DNA bands were generated through RAPD-PCR with the primer OPQ16. The molecular analysis employing both techniques revealed strong similarities between the DNA band patterns of the isolates belonging to the same Candida species. For the primer M13, eight isolates of Candida spp. with 100% band similarity were obtained, while with the primer OPQ16, 11 isolates were obtained with 100% band similarity (Figure 1).

The M13-PCR fingerprinting identified 100% similarity between two C. tropicalis strains from prawn (n=1) and sediment (point 3, n=1); four C. famata strains from prawns (n=2) and surface water (points 1 and 3; n=2) and two C. ciferrii strains from prawns (n=1) and sediment (point 1, n=1). In turn, the RAPD-PCR with the primer OPQ16 allowed identifying 100% similarity between two C. guilliermondii strains from surface water (points 1 and 4); five C. famata strains, two from prawn (n=1) and surface water (point 1, n=1) and three from surface water (points 1 and 2); two strains of C. parapsilosis from prawn (n=1) and surface water (point 4, n=1) and two C. ciferrii strains from prawn (n=1) and sediment (point 1, n=1) (Figure 1).

DISCUSSION

This study demonstrated the similarity among Candida spp. isolated from wild-harvested prawns and the aquatic environment where the animals inhabit, including surface water and sediment. The molecular analysis through M13-PCR fingerprinting and RAPD-PCR with OPQ-16 allowed evaluating this correlation, since these techniques generated varied band patterns among different Candida species and similar ones within the same species, thus presenting desirable and reliable results. In the present study, the primer OPQ16 was used to complement the results obtained through the M13-PCR fingerprinting and it generated a greater variety of DNA bands and identified a greater number of strains with 100% of similarity.

The recovered Candida species were simultaneously isolated from prawns and the aquatic environment and some of these isolates presented 100% similarity, even when recovered from different collection points. Thus, it was demonstrated that M. amazonicum contains in its gastrointestinal tract a representative cross-section of Candida spp. that colonize the water and the substrate where they live.

In addition, three sets of azole resistant strains were observed among the isolates from prawns and aquatic environment that presented 100% similarity. These findings are in accordance with those of BRILHANTE et al. (2011), who observed that 28.6% of the Candida spp. recovered from the intestinal tract of wild-harvested M. amazonicum isolated from Catu Lake were resistant to azole antifungals. Considering that the main mechanism of azole resistance among Candida spp. is the overexpression of efflux pumps (FENG et al., 2010), which is possibly related to the exposure of these microorganisms to chemical compounds, as an unspecific mechanism of cellular detoxification (JUNGWIRTH & KUCHLER, 2006), we strongly believe that Candida spp. could be used as indicators of environmental pollution, through the phenotypical assessment of their in vitro susceptibility profile.

Crustaceans accumulate pollutants in their tissues, such as hydrocarbons, pesticides and heavy metals (YILMAZ & YILMAZ, 2007), which might increase the azole resistance rate among yeasts from the microbiota, due to the overexpression of efflux pumps (KEENAN et al., 2007; MULLER et al., 2007), as a consequence of the chronic exposure to these chemical compounds. In this context, the use of this prawn as a sentinel for the isolation of Candida spp. seems potentially advantageous.

CONCLUSION

In conclusion, based on the obtained results, the use of M. amazonicum as a sentinel for the isolation of Candida spp. from aquatic environments is an interesting alternative for evaluating the environmental quality, considering that these animals harbor yeasts from the environment in their gastrointestinal tract. Additionally, due to their capacity to accumulate chemical pollutants in their tissues, they simulate the environmental conditions to which these yeasts are exposed, potentially contributing for monitoring the presence of resistant Candida spp. in the environment.

http://dx.doi.org/10.1590/0103-8478cr20140016

ACKNOWLEDGEMENTS

This research was supported by grants from the National Council for Scientific and Technological Development (CNPq; Brazil; Processes 302574/2009-3, 481614/2011-7, 504189/2012-3) and the Coordination Office for the Improvement of Higher Education Personnel (CAPES/PNPD 2103/2009, AE1-0052-000650100/11, Casadinho/PROCAD 552215/2011-2).

REFERENCES

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Raimunda Samia Nogueira Brilhante (I)* Manoel de Araujo Neto Paiva (I,II) Celia Maria de Souza Sampaio (II) Carlos Eduardo Cordeiro Teixeira (I) Joyce Fonteles Ribeiro (I) Debora de Souza Collares Maia Castelo-Branco (I) Tereza de Jesus Pinheiro Gomes Bandeira (I,III) Andre Jalles Monteiro (IV) Rossana de Aguiar Cordeiro (I) Jose Julio Costa Sidrim (I) Frederico Ozanan Barros Monteiro (V) Jose Luciano Bezerra Moreira (I) Marcos Fabio Gadelha Rocha (I,II)

(I) Departmento de Patologia e Medicina Legal, Faculdade de Medicina, Programa de Pos-graduacao em Microbiologia Medica, Centro Especializado em Micologia Medica (CEMM), Universidade Federal do Ceara (UFC). Rua Coronel Nunes de Melo, s/n, Rodolfo Teofilo. 60430-270, Fortaleza, CE, Brasil. E-mail: brilhante@ufc.br. * Autor para correspondencia.

(II) Faculdade de Veterinaria, Programa de Pos-graduacao em Ciencias Veterinarias, Universidade Estadual do Ceara (UECE), Fortaleza, CE, Brasil.

(III) Faculdade de Medicina, Centro Universitario Christus (Unichristus), Fortaleza, CE, Brasil.

(IV) Departmento de Estatistica e Matematica Aplicada, UFC, Fortaleza, CE, Brasil.

(V) Faculdade de Medicina Veterinaria, Programa de Pos-graduacao em Saude e Producao Animal da Amazonia, Universidade Federal Rural da Amazonia (UFRA), Belem, PA, Brasil.

Received 01.09.14 Approved 04.14.14 Returned by the author 07.24.14 CR-2014-0016.R2

Table 1--Species, access number, origin, isolation period and
antifungal susceptibility of 27 Candida spp. isolates used for
molecular analysis.

Species              Access number      Origin       Collection
                                                       point

C. famata            CEMM 1-1-259    Sediment        Point 3
C. guilliermondii    CEMM 1-1-260    Surface Water   Point 4
C. guilliermondii    CEMM 1-1-261    Sediment        Point 4
C. famata            CEMM 1-1-262    Sediment        Point 3
C. guilliermondii    CEMM 1-1-263    Prawn           Point 1
C. famata            CEMM 1-1-264    Prawn           Point 1
C. famata            CEMM 1-1-265    Prawn           Point 1
C. parapsilosis      CEMM 1-1-266    Surface Water   Point 4
C. famata            CEMM 1-1-267    Surface Water   Point 1
C. parapsilosis      CEMM 1-1-268    Prawn           Point 1
C. ciferrii          CEMM 1-1-269    Surface Water   Point 3
C. ciferrii          CEMM 1-1-270    Surface Water   Point 1
C. ciferrii          CEMM 1-1-271    Sediment        Point 4
C. ciferrii          CEMM 1-1-272    Sediment        Point 1
C. ciferrii          CEMM 1-1-273    Prawn           Point 1
C. famata            CEMM 1-1-274    Surface Water   Point 3
C. famata            CEMM 1-1-275    Surface Water   Point 3
C. famata            CEMM 1-1-276    Surface Water   Point 1
C. famata            CEMM 1-1-277    Prawn           Point 1
C. famata            CEMM 1-1-278    Surface Water   Point 4
C. parapsilosis      CEMM 1-1-279    Surface Water   Point 1
C. tropicalis        CEMM 1-1-280    Prawn           Point 1
C. tropicalis        CEMM 1-1-281    Sediment        Point 3
C. guilliermondii    CEMM 1-1-282    Surface Water   Point 4
C. guilliermondii    CEMM 1-1-283    Sediment        Point 3
C. guilliermondii    CEMM 1-1-284    Surface Water   Point 4
C. guilliermondii    CEMM 1-1-285    Surface Water   Point 1

Species                 Period       Resistance *

C. famata            October 2011    FLC/ITC
C. guilliermondii    November 2011   ITC
C. guilliermondii    November 2011   S
C. famata            November 2011   S
C. guilliermondii    December 2011   S
C. famata            November 2011   S
C. famata            November 2011   ITC
C. parapsilosis      November 2011   S
C. famata            August 2011     ITC
C. parapsilosis      March 2011      S
C. ciferrii          October 2011    S
C. ciferrii          August 2011     ITC
C. ciferrii          November 2011   S
C. ciferrii          October 2011    FLC/ITC
C. ciferrii          October 2011    FLC/ITC
C. famata            November 2011   S
C. famata            May 2011        S
C. famata            August 2011     FLC/ITC
C. famata            December 2011   S
C. famata            October 2011    FLC
C. parapsilosis      May 2011        S
C. tropicalis        October 2011    FLC/ITC
C. tropicalis        October 2011    FLC/ITC
C. guilliermondii    December 2011   S
C. guilliermondii    December 2011   S
C. guilliermondii    August 2011     ITC
C. guilliermondii    December 2011   FLC/ITC

* All strains were susceptible to amphotericin B, S: susceptible to
all tested antifungal drugs (amphotericin B, fluconazole and
itraconazole); FLC: resistance to fluconazole; ITC: Resistance to
itraconazole.
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Author:Brilhante, Raimunda Samia Nogueira; Paiva, Manoel de Araujo Neto; Sampaio, Celia Maria de Souza; Tei
Publication:Ciencia Rural
Date:Nov 1, 2014
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