Evaluation of the Antifungal and Antiyeast Activities from Recently

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 Actinobacteria (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 Streptomyces 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- Streptomycetaceae 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 bacteria 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 taxonomy 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 pathogenic 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 minutes 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

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