RESEARCH

Evaluation of the Antifungal ISSN No 2230-7885 CODEN JPBSCT and Antiyeast Activities NLM Title J Pharm Biomed Sci from Recently Isolated Erika Teresa Quintana1*, Diana Andrea Gil-Rivera1, Streptomycetes Amanda Alejo-Viderique1, Oliver López-Villegas1, ABSTRACT Luis Ángel Maldonado2,3

The indisputable role of (aka “actinomycetes”) in the pharmaceutical 1 Escuela Nacional de Ciencias Biológicas industry is represented by its innate capability to produce antibiotics, but little is known (ENCB), Instituto Politécnico Nacional (IPN), about the antifungal compounds they could also be producing. Among the Actinobacteria, México DF, México the genus produces antagonistic molecules against several pathogenic fungi and search/discovery programs should be revisited due to novel and/or poorly 2 Facultad de Química, Universidad Nacional studied fungal emerging diseases in humans and plants. In humans, this is certainly Autónoma de México (UNAM), México DF, important because novel antifungal therapies are among the most challenging problems México in intensive care medicine. The present study focused on the isolation and identification of 3 Rectoría – Secretaría General, Universidad novel organisms of the genus Streptomyces and their evaluation for antifungal activities. One hundred actinomycetes were isolated from Mexican soil samples and identified by Autónoma Metropolitana (UAM), México using two pairs of specific primers: (a) a pair of primers for the class Actinobacteria and DF, México (b) for the family ; all the isolates selected were found to contain LL-  Address reprint requests to A pm in their cell walls, a -wall chemical marker for . The isolates were then 2 Streptomyces *E. T. Quintana, Escuela Nacional de Cien- assigned to 38 multimembered groups on the basis of their morphological properties cias Biológicas (ENCB), Instituto Politécnico and one representative of each subgroup tested on antibiosis methods for in vitro Nacional (IPN), México DF, México antifungal–antiyeast activities. 97.4% and 81.6% of the isolates showed activity against E-mail: [email protected], the type strains of Aspergillus niger and Candida albicans, respectively. 16S rRNA gene sequencing of five isolates showed that they are closely related among each other and [email protected] felt in the subclade which is highly heterogeneous; the isolates Streptomyces griseus  Article citation: Quintana ET, Gil-Rivera may well represent novel species showing both antifungal and antiyeast activities, a DA, Alejo-Viderique A, López-Villegas O, property not fully explored for members of that 16S rRNA gene subclade. Streptomycetes Maldonado LA. Evaluation of the antifungal and antiyeast activities from recently KEYWORDS Actinomycetes, Streptomyces, antifungal activities, Aspergillus niger, isolated Streptomycetes. J Pharm Biomed Candida albicans Sci 2015;05(11):867–876. Available at www.jpbms.info Statement of originality of work: The INTRODUCTION manuscript has been read and approved by all the authors, the requirements for authorship have 1 The genus Streptomyces was proposed by Waksman and Henrici in 1943 on been met, and that each author believes that the the basis of morphological and cell-wall composition. Members of this manuscript represents honest and original work. genus are aerobic, gram-positive that have a high DNA G+C content Source of funding: ETQ CONACyT Jóvenes (%mol), contain LL-A2pm in their cell wall and lack characteristic sugars Talentos SIP-2015-RE/057-CONACYT (cell-wall type I) according to Lechevalier and colleagues2. Streptomycetes C110/2015 and SIP20150292. produce substrate mycelium, an extensively branched aerial hyphae with Competing interest / Conflict of interest: characteristic long chains of arthrospores that are formed in the aerial myce- The author(s) have no competing interests for 3–4 lia . The genus Streptomyces currently includes almost 600 validly described financial support, publication of this research, species and it is the foremost genera among the class Actinobacteria5. A patents, and royalties through this collaborative 16S rRNA gene sequences phylogenetic Streptomyces tree results in unique research. All authors were equally involved in clades that corresponds well with previous numerical studies3,6. discussed research work. There is no financial Phylogenetic relationships are important to establish because of the innate conflict with the subject matter discussed in the ability of Streptomyces to produce either antibacterial compounds7,8 or anti- manuscript. fungal–antiyeast molecules9–11. Members of the genus Streptomyces have the Disclaimer: Any views expressed in this unique capacity to produce many natural compounds with a wide range paper are those of the authors and do not of biological activities such as antimicrobials, antitumor agents, antiviral reflect the official policy or position of the Department of Defense. compounds, enzymes, enzyme inhibitors, growth promoters of animals and plants, immunomodulator agents and toxins12–15. Although the range of biological activity by the molecules they produce is extremely vast, the Streptomycetes have also shown to be antagonistic to different genera of patho- genic fungi but this area has received little or no attention when compared against the search for antibacterial compounds16–18.

Copyright © 2015

Received Date: 08 September 2015 – Accepted Date: 06 November 2015 – Published Online: 16 November 2015 868 E. T. Quintana

Since the discovery of actinomycin, Streptomycetes have malt extract agar (GYM27) plates until pure cultures were provided many significant biocompounds of high com- obtained; the plates incubated for 7 days at the condi- mercial value and continue to be routinely screened for tions mentioned above. Spore suspensions and microbial new bioactive substances, notably antimicrobial agents. biomass were cryo-preserved at −20°C in 20% glycerol Approximately two-thirds of all the naturally occurring (w/v) for long-term maintenance. antibiotics have been isolated from Actinobacteria and 13 about 75% are solely produced by Streptomycetes . Sadly, Colour grouping little is known about the ability of the Streptomycetes on the production of antifungal compounds. This is import- Putative Streptomycetes were subcultured onto GYM plates ant because of the significantly increasing number of incubated for 7 days at 30°C and checked for purity by opportunistic fungal infections in the past decades19. microscopic examination. Groups based on the mor- Nowadays, it is well documented that invasive fungal phology of the organisms were formed on the basis infections are among the most challenging problems of colour of the colony, colour of the spore mass and in intensive care medicine20,21.The most predominant absence/presence of diffusible pigment(s). fungi pathogens are the yeast Candida albicans and the fil- 22 amentous fungi of the genera Aspergillus and Fusarium . Determination of the diaminopimelic Furthermore, the increase in resistance of human–patho- acid (A pm) gen populations to known antimicrobials or antigungals 2 is a primary concern to the medical community and Extraction of the A2pm from whole-organism hydroly- pharmaceutical industry. Original sources and strategies sates of the isolates was performed following the pro- are urgently required to selectively find novel antimicro- cedure previously described by Staneck and Roberts28 bials or antifungals agents from putative new Streptomycetes albeit slight modifications. Briefly, two loops of fresh because recent reports suggest that they still remain an biomass (10 days growth) were suspended in 500 µL important source of bioactive compounds with antimi- of HCl 6N containing 100 µg of glass beads (<106 µm crobial activitiesthat could also be used in applied to agri- diameter; Sigma-Aldrich, Toluca, Mexico). The suspen- cultural, medicinal and veterinarian sciences. sions were then homogenised in a vortex for 5 min- utes and heated in an oven at 100°C for 4 hours. After MATERIALS AND METHODS centrifugation (5000 rpm, 5 minutes), the supernatant was transferred to a new clean Eppendorf tube and let Isolation of microorganisms to dryness in a multiblock heater at 100°C (Barnstead International, Dubuque IO USA). One millilitre of dis- Actinobacteria were isolated from five different arid soil tilled water was added (twice) to reconstitute each of the samples collected in Fresnillo, Zacatecas, Mexico; full hydrolysates. The final volume was 100 µL and 3–4 µL of details of the soil samples have been mentioned previ- each sample was used for a thin-layer chromatography ously23. The samples were coded as MX-1, MX-2, MX-3 (TLC) analysis. A TLC plate was developed in a saturated and MX-4. One gram of each soil was dried at room glass tank containing a methanol:water:6N:pyridine temperature. Each gram was added separately to 9 mL of (80:26:4:10, v/v) mixture for 4 hours or until the sol- isotonic solution (0.85% NaCl, w/v). The resultant dilu- vent reached ~2 cm before the top of the plate. The plate tion (10−1) was shaken for 30 minutes in a tube rotator was air-dried, sprayed with freshly prepared ninhydrin model SB2 at fixed speed (Fischer Scientific, Pittsburgh (0.2%, w/v dissolved in acetone; both Sigma-Aldrich, PA USA) and subsequently heated at 55°C for 5 min- Toluca Mexico) and heated at 100°C for 3 minutes. utes in a water bath (Grant Instruments, Cambridge Ltd Shepreth Cambridgeshire United Kingdom). Serial Evaluation of the biological activity against dilutions of each treated sample were made until it fungi and yeast reached the 10−3 dilution and 100 µL of each dilution were used to inoculate two different medias; (a) glucose Fresh cultures of Aspergillus niger ATCC 16404T and C. albi- yeast extract agar (GYEA24) supplemented with rifam- cans ATCC 10231T were used to test the colour-grouped picin and nystatin (5 and 50 μg/mL, respectively) and Streptomycetes. Solutions for C. albicans ATCC 10231T and for (b) humic acid vitamins agar (HV25,26) supplemented all the Streptomycetes were prepared according to a McFarland with nalidixic acid (50 μg/mL). Isolation plates were Nefelometer tube no. 5 (i.e. 15 × 108 CFU/mL). A incubated at 30°C for 4 weeks but checked each week. spore suspension following the procedure reported by Each isolation plate was observed by eye for colo- Duangmal et al.23 was prepared for A. niger ATCC 16404T. nies having a typical morphology of Streptomyces accord- All bioassays were carried out in Mueller Hinton (MH) ing to Goodfellow et al.5 Counts of Actinomycetes were agar plates (Difco, Mexico City, Mexico). Three different expressed as the mean number of colony-forming techniques were used to test the antifungal/antiyeast units (c.f.u) per gram of dry weight soil. Dry weights activity of each Streptomycetes: the streak technique for both were obtained by drying the soil samples at 105°C. The fungi and yeast, the eight-point and four-point tech- selected colonies were subculture onto glucose yeast niques only to detect any antifungal activity.

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The streak technique 30°C to allow growth, sporulation, production and dif- fusion of metabolites from the evaluated Streptomycetes into Fifteen microliters of each isolate solution were inoc- the MH media. After 7 days, 7 µL of the spore suspen- ulated and dispersed in 2 cm of the left side of a Petri sion from a fresh culture of A. niger ATCC 16404T were dish shown in Fig. 1A. Petri dishes were then incubated placed in the middle of the Petri dish at slightly less than for 7 days at 30°C to allow growth, sporulation, pro- 3 cm from each Streptomycetes point (Fig. 3A). The plates duction and diffusion of metabolites from the evaluated were now incubated for an additional 7 days at 30°C and Streptomycetes into the MH media. After 7 days, 7 µL of a again checked visually. A positive score for the Streptomycetes T suspension from fresh C. albicans ATCC 10231 biomass against the fungi was considered when no A. niger ATCC was spread out in perpendicular position 5 cm (right to 16404T growth was observed in the middle of the MH left) of the MH Petri dish growing the Streptomycetes (Fig. Petri dish (Fig. 3B–D) and after comparing against a con- 1A). Petri dishes were now incubated for an extra 7 days trol plate which contained no Streptomycetes (Fig. 3E). The and checked visually. A positive score for the Streptomycetes technique was modified from that described by Luna and against the yeast was considered when no C. albicans colleagues (2009) in order to evaluate the Streptomycetes. ATCC 10231T growth was observed in the middle of the MH Petri dish (Fig. 1B) and after comparing against a control plate which contained no Streptomycetes (Fig. 1C). The four equidistant point technique The same procedure was carried out using the A. niger Seven microlitres of each isolate suspension were inoc- T ATCC 16404 spore suspension as shown in Fig. 2A–C. ulated in four equidistant points almost to the border of the MH Petri dish as shown in Fig. 4A. The plates were The eight-point technique29 incubated as mentioned previously for 7 days and then 7 µL of the spore suspension from the fresh culture of A. Seven microlitres of each isolate suspension were inocu- niger ATCC 16404T was placed in the center of the Petri lated in eight equidistant points from the center of a Petri dish at slightly less than 5 cm from each Streptomycetes dish (i.e. 3 cm; Fig. 3A) and then incubated for 7 days at point (Fig. 4A). The plates were now incubated for

Fig. 1 A–C: The streak technique for evaluation of anti-Candida albicans ATCC 10231T activity (see Materials and Methods, The streak technique section for full details). The red arrow indicates the yeast position in the Petri dish; numbers 1–5 were either gram-positive or gram-negative bacteria.

Fig. 2 A–C: The streak technique for evaluation of the activity against Aspergillus niger ATCC 16404T (see Materials and Methods, the streak technique section for full details).

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Fig. 3 A–E: The eight-point technique (modified from Luna et al.29) for evaluation of the activity against Aspergillus niger ATCC 16404T [see Materials and Methods, the eight-point technique (Luna et al.29) section for full details].

Fig. 4 A–D: The four-point technique for evaluation of the activity against Aspergillus niger ATCC 16404T (see Materials and Methods, the four-point technique section for full details).

J Pharm Biomed Sci | Vol. 05 No. 11 | 867–876 Streptomycetes (Actinobacteria) applications 871 another 7 days and again checked visually as mentioned complete 16S rRNA gene sequences were compared against previously for the other techniques. A positive score for public databases using the BLAST option of the GenBank the Streptomycetes against A. niger ATCC 16404T was con- web site (http://www.ncbi.nlm.nih.gov). This provided the sidered if minimal fungal growth was observed radi- closest phylogenetic neighbours for each isolate and a phy- ally (Fig. 4B, C) after comparing against a control plate logenetic tree constructed using the neighbour-joining34 which contained no Streptomycetes (Fig. 4D). tree-making algorithm. The unrooted tree topologies were Despite the techniques used, controls of fungal and evaluated by bootstrap analyses35 of the neighbour-joining yeast growth were prepared separately and in duplicate. dataset after 1000 replicates. All plates were checked at least weekly up to 28 days. Inhibition (if present) was measured in millimeters (mm) RESULTS AND DISCUSSION and recorded in an Excel worksheet (Microsoft Corp, USA). One hundred colonies were chosen from the selec- DNA extraction and PCR amplification using tive isolation Petri dishes. The colonies showed typical class and family specific primers properties of Actinobacteria (most of them sporulated) and produced well-developed colonies when subcul- Genomic DNA for the putative Actinobacterial isolates tured onto fresh GYM plates. Once purified, colonies was extracted using a standard procedure reported previ- were grouped on the basis of morphological features ously30. DNA was used as a template for two different PCR such as colour of the colony, colour of the spore mass amplifications, the first one by using class Actinobacteria- and absence/presence of diffusible pigment(s) and 38 specific primers as described by Stach et al.31 and a second groups formed. A representative of each group was PCR by using the genus specific primers for the family chosen randomly for DNA extraction and for the pres- 32 Streptomycetaceae described by Monciardini et al. The PCR ence of A2pm in cell-wall hydrolysates by TLC analysis. All mixture was: 0.5 µL DNA template (final concentration of the chosen 38 isolates produced the expected single 100 ng µg−1), 5 µL 10X DNA polymerase buffer, 1.5 µL bands after two PCR amplifications, first confirming

MgCl2 (50 mM stock solution, Bioline, Taunton MA USA), their assignment to the class Actinobacteria (band size ca. 1.25 µL dNTPs (10 mM stock mixture, Bioline, Taunton 650 bp31) and second as part of the family Streptomycetaceae MA USA), 0.5 µL of each primer (20 µM stock solution), (band size ca. 600 bp32).Chemotaxonomic analyses of 2.0 units of Taq polymerase (Bioline, Taunton MA USA) TLC plates showed that the isolates presented whole-or- made up to 50 µL with deionised sterile distilled water. ganism hydrolysates rich in LL-A2pm, which then con- Amplifications were achieved using a Techne 512 ther- firmed its inclusion in the genus Streptomyces. Therefore, mocycler (Bibby Scientific, Stone Staffordshire United the random selection of putative Actinobacteria from the Kingdom) with slight modifications to the protocols orig- selective isolation Petri dishes was accurate and precise inally described by the authors31,32. PCR products were for Streptomycetes. The number of Actinobacteria recovered checked by horizontal electrophoresis (70 V, 45 minutes) were similar to those of Duangmal et al.23 which sug- on 1.0% agarose gels stained with ethidium bromide and gests that the chosen Mexican sample was indeed a good documented using an UVISave gel watch system (UVITEC source of Streptomycetes not only of those belonging to the Ltd, Cambridge United Kingdom). Streptomyces violaceoruber clade. An arbitrary set of values was used to track the anti- 16S RRNA amplification for species fungal and antiyeast activities coupled with a measuring identification and phylogenetic analysis in inhibition millimeters (mm) for each of the three techniques employed in this study. Under this arbitrary DNA from the isolates was also used as template for 16S scheme, a code level 1 corresponded to high (35–50 mm), rRNA gene amplification using the universal bacterial prim- a level 2 to moderate (20–34 mm), a level 3 to low (19–10 ers 27f and 1525r33. The PCR conditions have been described mm) and a level 4 to none inhibition activity (00–09 mm). elsewhere and the amplified PCR products (expected size The results for all the 38 representatives of each morpho- of 1500 bp) purified using a QIAquick PCR Kit (QIAGEN, logical group and how they were distributed with the Mexico City Mexico) following the manufacturer instruc- streak technique are shown in Tables 1 and 2. The results tions. PCR-purified products were sequenced at Macrogen were also employed to construct a graph that compared (Rockville MD USA) according to their instructions. Nearly the antifungal and antiyeast activity (Fig. 5).

Table 1 Number and percentage of isolates assigned to the different levels of inhibition. Level 1: High Level 2: Moderate Level 3: Low Level 4: None Antimicrobial activity against (50–35 mm) (34–20 mm) (19–10 mm) (09–00 mm) Aspergillus niger ATCC 16404T 30 (78.94%) 1 (2.63%) 4 (10.52%) 3 (7.89%) Candida albicans ATCC 10231T 12 (31.57%) 6 (15.78%) 7 (18.42%) 13 (34.21%)

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Table 2 Individual results for each of the 38 colour-grouped Streptomycetes. Inhibition values (mm) and level Candida Number of Code of the Level of Aspergillus niger albicans ATCC isolates isolates Level of inhibition inhibition ATCC 10231T 16404T 1 aislado-a 7 None 0 None 2 aislado-b 50 High 12 Low 3 aislado-d 41 High 19 Low 4 aislado-e 20 Moderate 18 Low 5 aislado-f 49 High 29 Moderate 6 aislado-g 35 High 19 Low 7 aislado-i 50 High 21 Moderate 8 aislado-k 50 High 20 Moderate 9 aislado-l 50 High 32 Moderate 10 aislado-m 50 High 18 Low 11 aislado-n 50 High 3 None 12 aislado-p 50 High 21 Moderate 13 aislado-q 50 High 18 Low 14 aislado-r 50 High 0 None 15 aislado-t 50 High 40 High 16 aislado-v 10 Low 0 None 17 aislado-w 50 High 38 High 18 aislado-x 50 High 36 High 19 aislado-y 50 High 39 High 20 aislado-z 50 High 39 High 21 aislado-aa 50 High 32 Moderate 22 aislado-ab 50 High 50 High 23 aislado-ae 50 High 35 High 24 aislado-af 50 High 43 High 25 aislado-ai 50 High 47 High 26 aislado-aj 50 High 47 High 27 aislado-al 50 High 38 High 28 aislado-añ 50 High 40 High 29 aislado-ba 13 Low 5 None 30 aislado-bb 50 High 5 None 31 aislado-bc 50 High 9 None 32 aislado-be 14 Low 5 None 33 aislado-bf 0 None 0 None 34 aislado-bh 50 High 0 None 35 aislado-bi 5 None 0 None 36 aislado-bj 50 High 3 None 37 aislado-bk 13 Low 0 None 38 aislado-bn 50 High 15 Low

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Fig. 5 A comparison of the antifungal and antiyeast activity for the 38 isolates using the streak technique.

No scale could be established for the eight-point a source of spirotetronate polyketides (chlorothricin) technique and only a third of the High-level strains which are useful in anticancer therapies38. In addition, against A. niger ATCC 10231T (i.e. aislado-d, aislado-i, aisla- the enanomycin group of antibiotics – which are also do-m, aislado-t, aislado-ab, aislado-af, aislado-ai, aislado-aj, aisla- produced by S. baarnensis – show very limited activity do-al and aislado-añ) were selected for further evaluation. against fungi and yeast39,40. S. fimicarius was reported to All of these strains showed similar levels of inhibition produce the griseorubin family of antibiotics which when compared to the results obtained with the first are only active against gram-positive, gram-negative, technique (data not shown). Again, the four-point tech- mycobacteria, mycoplasma and protozoa but not against nique reproduced the two previous techniques and the fungi or yeast41,42. S. flavofuscus was evaluated together highest antifungal activity was consistently observed with two closely related strains, namely S. albiaxilis and after 14 days when compared to the control plate. Isolates S. xinghaiensis, but again no activity against A. niger or aislado-ab, aislado-af, aislado-ai and aislado-aj showed slightly C. albicans was found43. No reports were found on the higher levels of inhibition than the rest of the evaluated antimicrobial activity of S. badius since this species has strains. Good congruence was found despite the tech- mostly been studied for B-lactamase genes and degrad- nique employed to evaluate the antimicrobial activity. ing enzymes44,45. Almost complete 16S rRNA gene sequences of 5 out Kang and colleagues10 reported on the selective of the 10 selected isolates were obtained and a BLAST isolation of Streptomycetes that show both antifungal and analysis showed that all the strains indeed belong to the antiyeast activities. However, none of their strains so far genus Streptomyces. A phylogenetic tree was constructed seem to be related to members of the S. griseus subclade with the 16S rRNA gene sequences of aislado-d, aislado-i, but, on the other hand, to species either related to S. aislado-j, aislado-m and aislado-af and the corresponding 16S violaceusniger or to any other species in the 16S rRNA gene rRNA gene sequences of several Streptomycetes. The result- Streptomycetes subclades. According to our results, it seems ing phylogenetic tree showed that the five Streptomycetes likely that antifungal and antiyeast Streptomycetes can also formed a distinct but well-defined subclade within be found in other Streptomycetes subclades, but the intrin- Streptomyces that is closely related to the S. griseus subclade sic complexity of the S. griseus subclade makes compar- (Fig. 6). Interestingly, the S. griseus subclade is formed by ative studies difficult among closely related strains or a subset of 31 species according to the study of Labeda even to unravel the relationship observed between the and colleagues6, located between the homogeneous16S isolates that were sequenced in this study. It is needed to rRNA gene sequencing clades 34 and 35 of such work6. extend the results presented in this work, for example Polyphasic studies have shed some light in the taxonomic to establish the accurate phylogenetic position of all the relationships between the 31 species which are pheno- isolates recovered and to unravel – if any – their intrinsic typically heterogeneous36. Such studies are important 16S rRNA gene relationship. because of the heterogeneity in the secondary metabolites Cole46 has critically reviewed several techniques for the members of the subclade produce but unpractical for testing biological compounds, including those with comparative studies with recently isolated strains6,36,37. antifungal activities. In his work, however, the assays for None of the four species selected to represent such antifungal activity refer to either already extracted or subclade in our phylogenetic tree (i.e. Streptomyces baarn- pure compounds. The approach that was followed in this ensis, S. badius, S. fimicarius and S. flavofuscus) have been asso- work combined both bits of the agar and paper diffusion ciated with the production of neither antifungal nor assays and was found to be very useful when search- antiyeast compounds. S. baarnensis has been reported as ing for antifungal–antiyeast activity from novel isolates

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Fig. 6 A partial 16S rRNA gene sequence phylogenetic tree showing the position of the most interesting isolates from this study.

without knowing the chemically active compound. From our point of view, the current prevalence of There is no doubt, though, that when several isolates fungal infections makes the evaluation of the antifun- are obtained, the techniques employed in this work can gal and antiyeast activities not only worth of more serve as a guide to further explore some isolates rather extensive studies given the innate ability of Streptomycetes than the whole lot of strains. It can be assumed, nev- to produce secondary metabolites but also because of ertheless, that the compounds produced by the tested the unexplored Actinobacterial diversity that could be Streptomycetes are aqueous-based compounds since they found in Mexican soils. The soil samples employed in diffused into the tested media; it is unlikely that the this study have been previously used as a source for S. compounds are of lipophilic origin. violaceoruber strains23 and now our study reports the pres- A Brasilian group evaluated a new coumarin deriva- ence of other Streptomycetes. It may even be worth looking tive against A. flavus and A. fumigatus both alone and cou- on the same soil sample for other Actinobacteria given pled to other antifungal drugs, namely, azoles47. The the unique ability of this bacteria to produce bioactive obvious next step regarding our work should defini- compounds. tively involve the elucidation of the chemical structure of the antifungal/antiyeast compounds produced by the ACKNOWLEDGEMENTS isolates in order to test the pure compound(s) according to the guidelines provided by Cole and/other authors, The main core of this work was awarded the “Best for instance, by bioautographic methods46,48. This would Biodiversity Poster” prize during the first Bergey’s also be of particular interest because of those isolates International Society for Microbial Systematics (BISMiS) that could have easily being assigned either to a higher meeting in Beijing China, 19–23 May 2011. This or lower level of antifungal activity on the basis of our work was entirely supported by grants to ETQuintana arbitrary mm cut-off point (e.g. aislado-e and aislado-g with (CONACyT Jóvenes Talentos Clave SIP-2015-RE/057- 20 and 35 mm, respectively) and to establish whether CONACyT C110/2015 and SIP20150292 and it is also all the Streptomycetes are producing the same or different part of the collaborative agreement between IPN-ENCB chemical compounds. and UNAM-ICML (UNAM No. 32939-2163-16-X-12).

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AAV acknowledges (a) CONACyT Master and Ph.D. fungal pathogens of turfgrass. World J Microb Biotechnol. degree Scholarships (No. 246636); (b) a CONACyT 1998;14:865–72. Jóvenes Talentos Scholarship in Jan and Feb 2013; 17. Jayamurthy H, Valappil Sajna K, Dastagar SG, Pandey A. Anti- (c) a PIFI-IPN Scholarship from August to December fungal potentials of extracellular metabolites of Western Ghats (2010-2015) and from Feb to Jun (2011-2015). DAGR isolated Streptomyces sp NII 1006 against moulds and yeasts. acknowledges the financial support of an IPN-PIFI and a Indian J Exp Biol 2014;52:1138–46. 18. Keikha N, Ayatollahi Mousavi SA, Nakhaei AR, Yadegari MH, TELMEX Program scholarships while doing the experi- Shahidi Bonjar GH, Amiri S. In vitro evaluation of enzymatic mental work of this manuscript. and antifungal activities of soil-actinomycetes isolates and their molecular identification by PCR. Jundishapur J Microbiol. REFERENCES 2015;8:e14874. 19. Arendrup MC. Candida and candidaemia. 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