Antibiofilm activity of Streptomyces toxytricini Fz 94 against Candida albicans ATCC 10231

Candida albicans is a significant cause of morbidity and mortality in immunocompromised patients worldwide. Biofilm formation by Candida species is a significant virulence factor for disease pathogenesis. Keeping in view the importance of Streptomyces' metabolites, the present study was initiated during the bioprospecting programme of Egyptian Streptomyces carried by the authors since 2013. Native Streptomyces isolates were recovered from soil samples collected from different governorates. Antifungal activity of forty isolates of Streptomyces were performed against planktonic (free cells) of C. albicans ATCC 10231 and resistant clinical Candida isolates. Streptomyces isolates showed high inhibition activity against free cells of Candida were further assayed against biofilm of C. albicans reference strain. The most active Streptomyces sp. (no.6) was identified phenotypically, biochemically and by using 16S rRNA. The 16S rRNA sequences obtained were compared with those deposited in the GenBank Database and registered with accession number KM052378 as S. toxytricini Fz94. Screening of S. toxytricini Fz94 extract capability in prevention and destruction of C. albicans reference strain biolfilm was assessed by resazurin dye adopted technique. In the pre-exposure scheme, the lowest concentration of 5 gL showed biofilm viability inhibition of 92% after 120 min, while Ketoconazole® gave 90 % inhibition at concentration of 2 gL. In post exposure, the concentration of S. toxytricini Fz94 extract 7gL caused 82 % inhibition of biofilms viability after 120 min, while Ketoconazole did not show any destruction capability. The cytotoxicity of S. toxytricini Fz94 crude extract results showed that it was nontoxic at 10 gL. S. toxytricini Fz94 is maintained in the Fungarium of Arab Society for Fungal Conservation (ASFC) with accession number FSCU-2017-1110. Key words– Bioprospecting – Egypt – Ketoconazole – Streptomyces toxytricini Fz94 –resazurin. Introduction Microorganisms growing in a biofilm are associated with chronic and recurrent human infections (Sutherland 2001). The most important feature of biofilm growth is the high resistance to antimicrobial agents that can be up to 1000-fold greater than that of planktonic cells (Amorena et al. 1999; Saginur et al. 2006; Hassan et al. 2011). The preferred life-style for most of microorganisms is biofilm rather than planktonic cells (Douglas, 2003). The consequences of mature biofilm development result in structural and phenotypic changes that has numerous Microbial Biosystems 2(1): 26–39 (2017) ISSN 2357-0334 http://fungiofegypt.com/Journal/index.html Microbial Biosystems Copyright © 2017 Sheir and Hafez Online Edition ARTICLE


Introduction
Microorganisms growing in a biofilm are associated with chronic and recurrent human infections (Sutherland 2001).The most important feature of biofilm growth is the high resistance to antimicrobial agents that can be up to 1000-fold greater than that of planktonic cells (Amorena et al. 1999;Saginur et al. 2006;Hassan et al. 2011).The preferred life-style for most of microorganisms is biofilm rather than planktonic cells (Douglas, 2003).The consequences of mature biofilm development result in structural and phenotypic changes that has numerous benefits to microbial cells that may include coherent attachment to surfaces, colonization of host tissues, the expression or enhancement of virulence traits, protection from stresses, sequestering of nutrients, enhanced cell-to-cell communication, dispersal, and survival during harsh conditions (Harding et al., 2009).However, Candida albicans is an important nosocomial pathogen in immunocompromised patients that has a high morbidity and mortality rate (Wisplinghoff et al., 2004, Mensa et al., 2008,).This is due to many of these C. albicans are implant-associated infections, owing in part to its greater capacity to form biofilms that are found on the surfaces of devices including central venous catheters, catheters, prosthetic heart valves, endotracheal tubes, pacemakers and joint replacements that cause bloodstream infection (Douglas, 2002, Chandra et al., 2005, Sardi et al., 2013).
To study Candida biofilms, several microtiter plate-based assays have been used recently to determine biofilm mass (Sherry et al., 2014), microbial physiological activity (Van den Driessche, et al., 2014, Noumi et al., 2010;De Logu et al., 2005, LaFleur et al., 2006), and extracellular matrix (Peeters et al., 2008).Their final results are based either on absorbance or fluorescence intensity at a certain wavelength, which means that fast and quantitative analyses are obtained with a microplate reader (Azevedo et al., 2009).These assays also show a broad applicability and reproducibility for many microorganisms (Peeters et al., 2008).
Recently, several microtiter plate-based assays have been used to study the Candida biofilms e.g.determination of mass (Sherry et al., 2014), microbial physiological activity (De Logu et al., 2005;LaFleur et al., 2006;Noumi et al., 2010;Van den Driessche et al., 2014), and extracellular matrix (Peeters et al., 2008).Their final results are based either on absorbance or fluorescence intensity at a certain wavelength, which means that fast and quantitative analyses are obtained with a simple microplate reader (Azevedo et al., 2009).These assays also show a broad applicability and a high repeatability for many microorganisms (Peeters et al., 2008).
The aim of this study was to evaluate the inhibitory activity of S. toxytricini Fz94 against planktonic cells of both reference and clinical isolates of C. albicans in vitro Moreover, its activity against biofilm of reference C. albicans in both prevention and destruction modes in comparison with standard antifungals.Finally, evaluation the cytotoxicity of S. toxytricini Fz94 against Human Lung (HL) epithelial cells was assessed.

Microorganisms
Isolation of Streptomyces Taxa of Streptomyces were isolated from subsurface (5-7 cm) soil samples collected from different governorates in Egypt from January 2013 till December 2015 using soil dilution-plate method (Kuster and Williams 1964) on starch casein agar media (Shirling and Gottlieb 1966).

Isolation of clinical Candida
Clinical samples (urine, sputum, and pus, vaginal and pleural fluids) were collected from Kasr Elini Hospital, Egypt for isolation of clinical Candida using standard Mycology methods (Odds and Bernarets 1994).Candida spp.were inoculated on CHROM agar and incubated at 37°C for 24 hrs.C. albicans was identified by type and colour of the colonies on CHROM agar media as light green (Devi and Maheshwari 2014).

Reference C. albicans
The reference C. albicans was obtained from American Type Culture Collection (ATCC) under reference number of 10231.

Identification of the bioactive Streptomyces isolate Phenotypic characterization
The Streptomyces strain was inoculated on Starch Casein Agar (SCA) medium, incubated at 30 °C for 5 days and the macro-morphology of strains including colors of mature sporulating aerial and substrate mycelia were monitored.However, the micro-morphology of the strain including the form of the sporophores was examined under light microscope.Furthermore, the morphology of the spores and the spore chain was investigated by Scanning Electron Microscope (SEM) (JEOL, JSM-5910, Japan).For the SEM, A plug of sporulating Sterptomyces sp.6 incubated for 7 days at 28 C on SCA medium was removed and fixed in glutaraldehyde vapor (2% v/v) at room temperature (RT) for 3 h.Then dehydrated through a series of ethanol solution (50, 60, 70, 80 and 95%, 15 min each; twice with 100% ethanol, 30 min/ time).Ethanol was substituted with acetone and subjected to critical-point dryer (CPD7510, Critical Point Drying/ Apparatus, Polaron, Rang) and then sputter coating with gold using a Gold Sputter (SPI-Module TM Sputter Coater, SPI Supplies, Division of Structure Probe Inc., USA) and observed by SEM (Hosny et al. 2014).

Physiological and Biochemical Characterization
Physiological characterization including melanin production was detected using International Streptomyces Project (ISP) included ISP-1, ISP-6 and ISP-7 media according to the method reported by Shirling and Gottlieb (1966), and carbon sources utilization was conducted as described in MacFaddin (2000) using each one of the nine sugars including D-glucose, Dsucrose, D-sorbitol, D-mannitol, L-arabinose, meso-inositol, L-rhamnose, D-melibiose and Damygdalin at a concentration of 0.5%.The biochemical tests including starch and gelatin hydrolysis, Voges-Proskauer, citrate utilization, indole, and H 2 S production were carried out according to MacFaddin (2000).Also, casein hydrolysis was performed as reported in Shirling and Gottlieb (1966) and urea hydrolysis was conducted as described by Chrisensen (1946).However, the results of biochemical and physiological properties of these isolates were confirmed by API ® biochemical identification system (bioMerieux, France).

Molecular identification DNA extraction
The extraction of genomic DNA was performed according to the protocol recommended for the DNA purification of Gene JET™ genomic DNA purification kit (Thermo Fisher Scientific) as follows: Streptomyces cells were harvested up to 2x10 9 in a 1.5 or 2 mL micro centrifuge tube, then the culture was centrifuged for 10 min at 5000 ×g and the supernatant was discarded.Eventually, the genomic DNA of lysed bacterial cells was precipitated with 20μL of Proteinase K Solution and purified using ethanol 70%.

Phylogenetic analysis
The alignment was manually adjusted and then the phylogenetic tree was constructed by using neighbour-joining (Saito and Nei 1987) in the MEGA program version 6 (Tamura et al. 2013).The confidence values of branches of the phylogenetic tree were determined using the bootstrap analyses based on 1000 resamplings (Felsenstien 1985).

Primary and secondary screening of Streptomyces isolates against reference strain of C. albicans
The primary and secondary screening were carried out against C. albicans (ATCC 10231) using cut plug method (Pridham et al. 1956) and agar well diffusion method (Kavanagh 1972) respectively after incubation at 37 °C for 24 h.

Screening for antifungal activity of Streptomyces isolates against clinical C. albicans isolates
A suspension of each of the clinical isolates plated onto CHROMagar Candida equivalent to 0.5 McFarland Standard was inoculated on Mueller-Hinton agar to which 2% glucose and 0.5 g mL -1 methylene blue dye was added (MH-GMB) using a sterile cotton-swab, removing excess fluid by pressing against the tube according to Tan and Peterson (2005) technique.

Screening of the antifungally active Streptomyces sp. against reference C. albicans biofilm using resazurin method Preparation of the reference C. albicans
Fresh cultures of reference C. albicans were inoculated in YPD broth and incubated overnight at 30ºC in orbital shaker of 200 rpm.Cells were harvested at 4000 rpm for 5 min, washed in sterile Phosphate-buffered saline (PBS), centrifuged, resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid (MOPS) (pH 7.0) to be counted by Bürker counting chamber and adjusted to a cellular density of 1×10 6 cells/mL for biofilm formation in 96-well microtitre plate (Ramage, 2001).

Effect of extract against C. albicans biofilm Prior-to-exposure (prevention mode)
The crude extracts were added in 2% concentration of total volume simultaneously with the Candida suspension in RPMI media.Ketoconazole (0.2 and 2 gL -1 ) and metronidazole (1 and 10 gL -1 ) were used as controls.Therefore, all compounds were dissolved in distilled water of 50 times of the desired concentration and were incubated under optimized conditions.After 24 h incubation, the medium was discarded and non-adherent cells were removed and 200 μL of resazurin was added in each well.

Post-exposure (destruction mode)
After biofilm formation for 24 h, the planktonic suspension was removed by aspiration using multichannel automatic pipette and replaced by fresh RPMI 1640 medium along with 2 % of crude extract.Also, 2% of ketoconazole and metronidazole were used as standard controls.The plates were incubated for further 24 h and then the medium was removed, the wells were washed with PBS and 200 μL of resazurin was added in each well.Eventually, The fluorescence was measured using an excitation filter of 560 nm and an emission filter of 590 nm using Varioskan Multimode Plate Reader (Thermo Fisher Scientific, Vantaa, Finland) after optimum incubation time in both prevention and destruction modes.

Cytotoxic evaluation of antifungal compounds on Human Lung HL epithelial cells
It was done using resazurin method according to Karlsson et al. (2012).Human Lung epithelial HL cells (Kuo and Grayston 1990) were grown in RPMI 1640 medium supplemented with 7.5% fetal bovine serum (FBS), 2 mM L-glutamine and 20 μg/mL gentamycin.For the cell viability assays, the cells were seeded into 96-well plates at density 60.000 cells/ well and incubated overnight before starting the exposure.The extracts were diluted with cell culture medium to yield the indicated concentrations, the medium from the 96-wellplates with cells was removed and aliquots of 200 μL/ well of the samples were added in three replicates.The plates were incubated for 24 h after which the samples were removed and wells were washed with 100 μL PBS.After washing, 20 μM resazurin solution in 200 μL PBS was added into each well and the plate was incubated at 37 ˚C for 2 h.The fluorescent signal (excitation/emission 560/590 nm) resulting from the reduced form of resazurin was read with Varioskan Multimode Plate Reader (Thermo Fisher Scientific, Vantaa, Finland).The results were expressed as viability percentages normalized using a negative control.

Data processing and statistical analysis
Statistical parameters included signal window coefficient Z-factor, signal-to-noise (S/N), signal-to-background (S/B) and separation band were calculated according to Zhang et al., (1999) and Bollini et al. (2002).In all equations, SD min , X min and SD max , X max represent the standard deviations and means of the minimal (min) and maximal (max) signals, respectively.The means of the relative fluorescence units (RFU) measurements of negative (RPMI 1640 medium, minimal signal, min) and positive (untreated biofilms, maximal signal, max) control wells, respectively.The hit limits were used in the screening experiments to distinguish active from non-active compounds (Sandberg et al. 2009).

Isolation of Streptomyces and Screening of antifungal activity against reference strain
Out of the forty Streptomyces isolates, Streptomyces sp.6 isolated from soil of cattle farms in Dakahlya showed strong antifungal activity was selected for identification.

Identification of the active Streptomyces isolate
Phenotypic, physiological and biochemical characterization Streptomyces sp.6 has buff color of the aerial mycelium.The substrate mycelium is unpigmented and no diffusible pigment was produced (Table 1).The microscopic examination illustrated that the spore chain morphology is long straight spore shaped (Fig. 1).Streptomyces sp.6 utilized starch as a carbon source.Streptomyces sp.6 utilized casein as a good nitrogen source and it was able to produce melanin.The biochemical characterization of Streptomyces sp.6 revealed that it has the capability to produce different enzymes such as arginine dihydrolase, gelatinase, utilized citrate and produced acetoin (Voges-Proskaur).

+
A plus sign (+), the isolate gave positive reaction for the test; a negative sign (-), the isolate gave negative reaction for the test.The isolate' metabolic activities was confirmed using API ® strips.

Screening of S. toxytricini Fz94 against clinical C. albicans isolates
In vitro antifungal susceptibilities of these isolates showed that almost all the clinical C. albicans were resistant to metronidazole and polymyxin B (93.3% & 86.6%) respectively and to a lesser extend resistant to fluconazole, nystatin and amphotericin B (60, 53.3 and 40%) respectively.On the other hand, 100% of clinical C. albicans isolates were susceptible to both crude extract S. toxytricini Fz94 and ketoconazole.

Resazurin test of antifungal activity of S. toxytricini Fz94 on biofilm forming reference C. albicans Optimization of C. albicans biofilm forming conditions
The optimal conditions for C. albicans ATCC 10231 biofilm formation, such as (volume of bacterial inoculum, growing time, static or shaking cultivation) and staining procedures (resazurin incubation time, elimination of washing steps).It was found that the optimal conditions for biofilm formation are: using 200µL of 1.0 × 10 6 cells per mL as inoculums for 24 h under shaking or static conditions.The resazurin assay optimal conditions were: incubation of resazurin for 90 and 120 min.

Effect of extract against C. albicans biofilm
Prior-to-exposure (prevention mode) In the pre-exposure scheme, at the lowest concentration of the extract of S. toxytricini Fz94 (5 gL -1 ) a 92% inhibition of biofilm viability after 120 min was detected, which is more potent than ketoconazole that gave 70 and 90% at concentrations of 0.2 and 2 gL -1 respectively.However, metronidazole that caused 25 and 30 % at concentrations of 1 and 10 gL -1 (Fig. 3).

Post-exposure (destruction mode)
In post exposure, the extract of S. toxytricini Fz94 (7 gL -1 ) caused 82 % inhibition of biofilms viability after 120 min, Metronidazole caused inhibition of 80% at concentration of 1 gL -1 and wasn't active at higher concentration, while ketoconazole didn't show any destruction ability.

Cytotoxic evaluation of crude extract of S. toxytricini Fz94 on Human Lung (HL) epithelial cells
To further characterize the biological activity of the crude extract of S. toxytricini Fz94, it was evaluated for its effect on human epithelial cells of S. toxytricini Fz94 was nontoxic at 10 gL -1 (>85% viability), while it showed toxicity at 20 gL -1 (15% viability).

Discussion
Biofilm formation is one of the most important virulent factor of Candida spp. that has led to high prevalence of Candida infections concomitant with high morbidity and mortality rate in hospitalized patients (Pfaller and Diekema 2007;Favero et al. 2011;Vinitha and Mamatha 2011).C. albicans ATCC 10231, the target test strain in this study, has been a widely used as reference strain for antifungal (Atta 2010;Saurav and Kannabiran 2012) and biofilm research (De Logu et al. 2005;Palmeira-de-Oliveiraa et al. 2012).However, the aim of this study was screening of antifungal agents from genus Streptomyces against reference strain and clinical Abbreviations R, Resistant; I, Intermediate; S, Sensitive; KET, Ketoconazole; FCA, Fluconazole; AB, Amphoteracin B; NS, Nystatin; MTZ, Metronidazole; PB, Polymxin B; FD, Fucidic acid.

% Biofilm viability
Concentration (gL -1 ) isolates of C.albicnas and thereafter, evaluates the potential of this antifungal crude extract in prior to exposure and post exposure of reference strain of C.albicans.toxytricini Fz94 extract in prevention (P) and destruction (D) modes after resazurin incubation times 90 and 120 min.Abbreviations: Ket, ketoconazole; Mtz, metronidazole; HL%, hit limit percent.Positive hits found are those scoring below the hit limit.
The screening studies of the forty Streptomyces isolates resulted in a potent Streptomyces sp. 6. that has been identified using classical methods including morphological, physiological, biochemical characterization using 16S rRNA as S. toxytricini (strain fz94) and was deposited in the GenBank nucleotide sequence database under accession numbers of KM052378.Nowadays, detection and classification of Streptomyces is also commonly performed by molecular approaches (Locatelli et al. 2002).However, S. toxytricini was recorded to produce pancreatic lipase inhibitor which is lipstatin of which the antiobesity drug orlistat is a derivative (Barbier and Schneider 1987;Weibel et al. 1987).Also, it has antifungal activity against C. albicans (Abdel Azeiz et al. 2016).
In these screening studies, S. toxytricini fz94 showed strong antifungal activity against C. albicans ATCC 10231 and all the clinical Candida isolates which was similar to ketoconazole.This result was in accordance with Ng et al. (2000) who reported the same activity of ketoconazole against clinical Candida isolates.It was selected for evaluation for its anti-biofilm activity against C. albicans ATCC 10231 using resazurin method.However, In recent years, biofilm quantification assays were based on microtiter plates using several probes such as XTT which was the most commonly used method (Ramage et al. 2011), crystal violet (CV) (Peeters et al. 2008), and resazurin to a lesser extent in case of Candida biofilms (Driessche et al. 2014).Resazurin assay has been proven to be simple, fast, cost-effective and suitable for highthroughput method for biofilm quantification (Sandberg et al. 2009).Therefore, it was essential to optimize the biofilm formation and resazurin incubation time before evaluation of antibiofilm activity of the crude extract.The optimal biofilm formation according to statistical performance detected using inoculum volume of 200 µL of 1.0 × 10 6 cells per mL cellular density of C. albicans concentration for 24 h at 37°C, 100 rpm which is in accordance with Ramage et al. (2001) results.Because resazurin is nontoxic, microorganisms continue duplicating during resazurin incubation.As such, baseline fluorescence rises proportionally with incubation; the best result was achieved after adding the resazurin by 120 min.Therefore, the fluorescence was measured after 90 and 120 min for more convenience.In the pre-exposure scheme, at the lowest concentration of S. toxytricini Fz94 (5 gL -1 ) extract caused 92% inhibition of biofilm viability after 120 min.Whereas, ketoconazole caused 70 and 90% biofilm inhibition at concentrations of 0.2 and 2 gL -1 respectively.However, metronidazole caused 25 and 30 % at concentrations of 1 and 10 gL -1 .In post exposure, the extract of S. toxytricini Fz94 (7 gL -1 ) caused 82 % inhibition of biofilms viability after 120 min.While ketoconazole didn't show any destruction ability.Results showed that the antifungal produced by S. toxytricini fz94 had anti-Candida biofilm activities in both prevention and destruction modes.However, search in the literature revealed that there is few literatures regarding anti-Candida biofilm from Streptomyces spp.such as Pierce et al. (2009) and Rajalakshmi et al. (2014) only in prevention mode.
It was important to study the effect of the bioactive extract on cell lines because of the predicted cytotoxicity of the antifungal metabolites.However, cytotoxicity was carried out against normal Human Lung epithelial HL cells (Kuo and Grayston 1990) using resazurin method (Karlsson et al. 2012).The crude extract of S. toxytricini fz94 showed that it was nontoxic at 10 gL -1 (>85% viability), while it showed toxicity at 20 gL -1 (15% viability).

Conclusion
Based on the data available out of this study is that S. toxytricini fz94 extract may represent effective and safe anti-Candida biofilm at concentrations in prevention and destruction modes using resazurin method.It was similar to ketoconazole against clinical Candida isolates and in prevention of Candida biofilm but crude extract of S. toxytricini was more potent than ketoconazole in destruction of C.albicans biofilm.Additionally, more chemical studies will be needed to isolate and identify the active principles of the extract S. toxytricini evaluated in the present study.

Fig. 3 -
Fig. 3-Comparison of percent control (viability) of biofilm using different concentrations of S.toxytricini Fz94 extract in prevention (P) and destruction (D) modes after resazurin incubation times 90 and 120 min.Abbreviations: Ket, ketoconazole; Mtz, metronidazole; HL%, hit limit percent.Positive hits found are those scoring below the hit limit.Fluorescence was measured at λ excitation =570 nm and λ emission =590 nm.

Table 1
The phenotypic, physiological and biochemical characteristics of the bioactive Streptomyces sp.6

Table 2
Antifungal activity of crude extract of S. toxytricini Fz94 and reference antibiotics against clinical C. albicans isolates