RESUMEN

El objetivo de este estudio fue determinar la actividad antimicrobiana de un cultivo de Streptomyces sp. 6E3 aislado de minerales frente a diferentes cepas patógenas, producir un extracto y estimar la concen tración mínima inhibitoria (CMI) de las fracciones contra Staphylococcus aureus resistente a meticilina (SARM). La cepa Streptomyces sp. 6E3 mostró actividad antimicrobiana principalmente contra Staphy lococcus aureus (S. aureus). Cinco de las seis fracciones presentaron actividad antimicrobiana y la más efectiva dio una CMI de 0,88 ug/mL frente a S. aureus ATCC 33862, 0,44 ug/mL frente a S. aureus ATCC 43300 y 1,76 ug/mL frente a S. aureus cepa SARM. Streptomyces sp. 6E3 tiene un potencial antimicrobiano frente a cepas de S. aureus resistentes a meticilina y no resistentes, siendo de interés la realización de más estudios sobre sus metabolitos activos.

Palabras clave:
Streptomyces; Minerales; Agentes Antiinfecciosos; Extractos Celulares; Bacteria; Staph ylococcus aureus; Productos Biológicos; Pruebas de Sensibilidad Microbiana

ABSTRACT

The objectives of this study were to determine the antimicrobial activity of a culture of Streptomyces sp. 6E3 isolated from minerals against different pathogenic strains, to produce an extract and to estimate the minimum inhibitory concentration (MIC) of the fractions against methicillin-resistant Staphylococ cus aureus (MRSA). Streptomyces sp. 6E3 showed antimicrobial activity primarily against Staphylococcus aureus (S. aureus). Five of the six fractions presented antimicrobial activity and the most effective gave a MIC of 0.88 ug / mL against S. aureus ATCC 33862, 0.44 ug / mL against S. aureus ATCC 43300 and 1.76 ug / mL vs. a S. aureus MRSA strain. Streptomyces sp. 6E3 has an antimicrobial potential against S. aureus strains resistant to methicillin and non-resistant, being of interest carrying out of more studies on its active metabolites.

Keywords:
Streptomyces; Minerals, Anti-infective agents; Cell extracts; Bacteria; Staphylococcus aureus; Biological Products; Microbial Sensibility Tests

INTRODUCTION

Antibiotic resistance is one of the most important threats to global health, food security and national development. Currently, different alternatives are being developed to combat microbial strains that present multi-resistance, and one of them is the search for new secondary metabolites with antimicrobial bioactivity.

Among the microorganisms that produce bioactive compounds, Streptomyces are the most important genus of bacteria and produces compounds such as polyketide, peptides and polyketide-peptide hybrids, all of which have been characterized with different biological activities as antibacterial, antifungal and anticancer ¹1. Sottorff I, Wiese J, Lipfert M, Preußke N, Sönnichsen FD, Imhoff JF. Different secondary metabolite profiles of phylogenetically almost identical Streptomyces griseus strains originating from geographically remote locations. Microorganisms. 2019;7(6). pii: E166..

Approximately 60% of all known antibiotics against Gram-positive and Gram-negative bacteria have been isolated from Streptomyces, including tetracycline, daptomycin and chloramphenicol ²2. Klementz D, Döring K, Lucas X, Telukunta KK, Erxleben A, Deubel D, et al. StreptomeDB 2.0-an extended resource of natural products produced by streptomycetes. Nucleic Acids Res. 2015;44(D1):D509-D14.. Lately, with the purpose of finding new metabolites with antimicrobial activity from these microorganisms (Streptomyces), the isolation of these bacteria has begun to be con ducted from little explored environments such as the sea, plants ³3. Liang L, Haltli BA, Kerr RG. Draft Genome Sequence of Streptomyces sp. Strain, an Antibiotic Producer Isolated from Marine Sediment in Prince Edward Island, Canada. Microbiol Resour Announc. 2019;8(35):e00870-19.^,44. Qi D, Zou L, Zhou D, Chen Y, Gao Z, Feng R, et al. Taxonomy and Broad-Spectrum Antifungal Activity of Streptomyces sp. SCA3-4 Isolated From Rhizosphere Soil of Opuntia stricta. Front Microbiol. 2019;10:1390. and even minerals ⁵5. Hurtado J, Pacheco SL, Sheen P, Ugarte D. Actinobacteria isolated from Mineral Ores in Peru. J Microbiol Biotechnol Food Sci. 2019;2019:366-70..

In Peru’s marine environments, species with antibacterial activity have been found in strains of methicillin-resistant Staph ylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis⁶6. León J, Aponte JJ, Rojas R, Cuadra D, Ayala N, Tomás G, et al. Estudio de actinomicetos marinos aislados de la costa central del Perú y su actividad antibacteriana frente a Staphylococcus aureus meticilina resistentes y Enterococcus faecalis vancomicina resistentes. Rev Peru Med Exp Salud Publica. 2011;28(2):237-46. and also a strain capable of acting against patho genic strains resistant to beta-lactams ⁷7. Navarro ML, León J, Huamán M, De Amat C, Morales M, Flores L, et al. Identificación de un Complejo Antimicrobiano Natural, producido por Streptomyces californicus 13A2 inhibidor de Bacterias B-Lactámicos Resistentes de Origen Hospitalario aisladas del Instituto Nacional Materno Perinatal de Lima, Perú. Vitae. 2014;21:S59..

This study aims to determine the antimicrobial activity of cultures and metabolic extracts of Streptomyces sp. 6E3 against different pathogenic enterobacteria, such as Staphylococcus aureus and Candida sp.

THE STUDY

Experimental study to analyze the antimicrobial activity of the Streptomyces sp. 6E3 strain. This strain was isolated from minerals and identified previously, phenotypically and genetically ⁵5. Hurtado J, Pacheco SL, Sheen P, Ugarte D. Actinobacteria isolated from Mineral Ores in Peru. J Microbiol Biotechnol Food Sci. 2019;2019:366-70.. The strains confronted with Streptomyces sp. 6E3 were Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 33862, Proteus mirabilis ATCC 12453, Salmonella typhimurium ATCC 25241, Shige lla sonnei ATCC 25931, Candida albicans ATCC 90028, Staphylococcus aureus ATCC 43300 (MRSA) and Staphylococcus aureus (MRSA), from Instituto de Medicina Tropical Alexander Von Humboldt (Lima).

The production phase was determined by means of the antimicrobial activity test, in which the double-layer method of Singh et al. was used. ⁸8. Singh LS, Baruah I, Bora T. Actinomycetes of Loktak habitat: isolation and screening for antimicrobial activities. Biotechnology. 2006;5(2):217-21.⁾ Cultures of Streptomyces sp. 6E3 sown in the center of the XGAL agar from 3, 5, 7 and 10 day growth plates were compared to strains of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 33862. During these growth stages, colony growth was observed as well, both microscopically and macroscopically.

We used the double layer method of Singh et al. to screen the antimicrobial activity of the strain ⁽⁸8. Singh LS, Baruah I, Bora T. Actinomycetes of Loktak habitat: isolation and screening for antimicrobial activities. Biotechnology. 2006;5(2):217-21.. The experiment was carried out twice against each pathogenic strain. For the extracts and fractions, the agar diffusion method modified by Rojas et al. was used ⁽⁹9. Rojas JJ, García AM, López AJ. Evaluación de dos metodologías para determinar la actividad antimicrobiana de plantas medicinales. Bol latinoam Caribe plantas med aromát. 2005;4(2):28-32..

Ethyl acetate solvent was used for the preparation of the extracts. Thirty XGAL plates were sown and, with the aid of a mi cropipette, 3 ml of the solvent was added to the culture. The cells moistened by the solvent and the supernatant were swept and collected in Falcon tubes. The content from the mentioned tubes was homogenized with ultrasonic agitation for 15 minutes at room temperature. The tubes with the lysed cells were centrifuged for 10 minutes at 5000 rpm. The supernatant was then removed and the ethyl acetate solvent, evaporated, first in a rotavapor with a pressure of less than 250 millibars and in a water bath at 40 °C, and then with a nitrogen gas stream, until 147 mg of extract was obtained, which was kept at -20 °C until use.

A reverse-phase thin-layer chromatography (TLC Silicagel 60 RP18 from Merck®) with a mobile phase of acetonitrile (water in a ratio of 2:1) was performed on this extract to observe the number of compounds under a UV lamp at 254 nm and 366 nm. In addition, an antibiogram was performed by the diffusion method with discs to observe whether the desired antimicrobial effect was still present.

We used a glass column packed with silica gel RP60 from Merck®, pre-washed with methanol for the extract fractionation. 50 mg of the extract were dissolved in 400 uL ethyl acetate. The mobile phases were 20 mL of acetonitrile and water in ratios of 1:1 (v/v), 1:2, 6:4, 7:3, 8:2, 9:1 and 10:0. The fractions were collected in test tubes up to a volume of 4 mL and were subjected to reverse-phase thin-layer chromatography using an acetonitrile mobile phase: water, in a ratio of 3:1.

The chromatographies were observed under UV light of 366 nm. The fractions that presented similar chromatographic profiles were collected and the solvent was removed by means of the rotary evaporator. Finally, six fractions named A, B, C, D, E and F were obtained. A thin layer chromatography and an antibiogram, according to the disc diffusion method, were con ducted again on the six fractions, to observe which of the fractions had the antimicrobial effect.

The method of Wiegan et al.¹⁰10. Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2008;3(2):163-75.⁾ was used to determine the minimum inhibitory concentration (MIC). As positive control, vanco mycin was used for MRSA strains, and penicillin for Staphylococcus aureus, which ranged from 120 to 0.06 ug/mL. The negative control was a Mueller Hinton broth inoculated with a standard strain; the blank one was a sterile broth. The six fractions were dissolved in 400 uL dimethyl sulfoxide (DMSO) to prepare stock solutions, and make the respective dilutions. The amounts of the fractions used to make the stock solutions were as follows: 0.5 mg of the B fraction; 0.5 mg of the C fraction; 0.5 mg of the D fraction; 2 mg of the E fraction; and 1 mg of the F fraction.

The microplate was incubated at 37 °C for 24 hours. The results were obtained by two methods: a) triphenyltetrazolium chloride was used, in which the color change of the microwell contents to red indicated microbial growth, and b) the micro plate was placed in an ELISA reader, in which only turbidity was measured. The MIC was found by means of absorbance versus concentration curves, according to the methodology of Devienne et al. ¹¹11. Devienne KF, Raddi MSG. Screening for antimicrobial activity of natural products using a microplate photometer. Braz J Microbiol. 2002;33(2):166-8.⁾

Descriptive statistical analysis was performed to analyze the results of antimicrobial activity tests by the double layer and disc diffusion method.

RESULTS

Antimicrobial activity was observed in the 7- and 10-day cultures, which was related to the macroscopic observation of a red dish substance in the colony, while microscopically spores were observed, indicating the end of its mycelial phase (Figure 1).

In screening with Staphylococcus aureus ATCC 33862, Staphylococcus aureus ATCC 43300 (MRSA) and Staphylococcus au reus clinical strain (MRSA), inhibition halos from 21.5 mm to 42 mm in diameter were observed. All other strains were found to have inhibition halos of 1.5 mm to 3 mm, so the three Staphylococcus aureus strains were continued.

An extract of 141.9 mg was obtained, from which six fractions named A, B, C, D, E and F were obtained and six chromato graphic spots numbered 1, 2, 3, 4, 5 and 6, respectively, could be identified. The thin layer chromatography performed on the fractions corroborated the presence of the same chromatographic spots present in the raw extract (Figure 2).

The antibacterial activity of the fractions was determined and inhibition halos of 8 to 15 mm were observed against Staphylo coccus aureus strains. The D fraction showed the best activity, as well as a lower MIC unlike the rest of the fractions (Table 1 and Figure 3). The A fraction did not show any activity with the disk diffusion method, which is why the MIC was not determined.

DISCUSSION

Bioactive compounds are mostly produced as secondary metabolites and some of them may also be pigments ¹²12. Rao MPN, Xiao M, Li W-J. Fungal and Bacterial Pigments: Secondary Metabolites with Wide Applications. Front Microbiol. 2017;8(1113).. Many Strep tomyces produce pigmented compounds with antimicrobial activity, such as the antibiotics actinorhodin (13) and roseoflavin ¹⁴14. Otani S, Takatsu M, Nakano M, Kasai S, Miura R. Roseoflavin, a new antimicrobial pigment from Streptomyces. J Antibiot (Tokyo). 1974;27(1):88-9.^). The reddish coloration observed in the seven-day cultures may be related to the production of some antimicrobial com pound produced by this bacterium.

The Streptomyces strain used in this investigation is of mineral origin, specifically from a concentrate of arsenopyrite ⁵5. Hurtado J, Pacheco SL, Sheen P, Ugarte D. Actinobacteria isolated from Mineral Ores in Peru. J Microbiol Biotechnol Food Sci. 2019;2019:366-70.. It is evident that mining environments are very important for the search of strains with potential bioactivity, the same has been demonstrated in a study, in which Pleurostomophora sp. isolated from minerals produced anti-inflammatory compounds ⁽¹⁵15. Stierle AA, Stierle DB, Girtsman T, Mou T, Antczak C, Djaballah H. Azaphilones from an Acid Mine Extremophile Strain of a Pleurostomophora sp. J Nat Prod. 2015;78(12):2917-23..

Using the double-layer method (Table 1) it was observed that the Streptomyces strain, although it showed inhibition of the gram-negative strains’ growth, greater activity was obtained against the group of gram-positive strains which were represented by the three strains of Staphylococcus aureus, including those resistant to methicillin. Streptomyces have been isolated from poorly studied environments, such as the marine environment. Strain RT 408, which produces a polycleotide ¹⁶16. Sujatha P, Raju KVVSNB, Ramana T. Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus. Microbiol Res. 2005;160(2):119-26., and coral associated Streptomyces¹⁷17. Ubillus JA, Quispe JL, Durán RR, Trujillo SM, Salazar LL. Actividad antimicrobiana y sinérgica de metabolitos producidos por Streptomyces erythrogriseus M1077 de origen marino. RSVM. 2015;35(1):13-9., which inhibits gram-positive strains, including MRSA, have been isolated.

The extraction process was carried out by the surface sweeping of the culture plate, and it is possible that method facilitated recovery of the metabolites released by the bacteria into the environment or the ones contained within the cells.

In the case of this strain, at least six chromatographic spots have been visualized by means of thin

layer chromatography (Figure 2). Many of the species of the genus Streptomyces produce more than one metabolite depending on the product ob tained, which is related to the solvent used and the carrying capacity according to its polarity and that of the compounds ¹⁸18. Ilic S, Konstantinovic S, Todorovic Z, Lazic M, Veljkovic V, Jokovic N, et al. Characterization and antimicrobial activity of the bioactive metabolites in streptomycete isolates. Mikrobiologiia. 2007;76(4):480-7.^,1919. Arasu MV, Duraipandiyan V, Agastian P, Ignacimuthu S. In vitro antimicrobial activity of Streptomyces spp. ERI-3 isolated from Western Ghats rock soil (India). J Mycol Med. 2009;19(1):22-8.. From the metabolites found, only some would have antimicrobial activity. Streptomyces coelicolor, in addition to producing

actinorhodin, also produces other compounds with antimicrobial activity ¹³13. Nodwell JR. Microbe Profile: Streptomyces coelicolor: a burlesque of pigments and phenotypes. Microbiology. 2019;165(9):953-5. and Streptomyces violaceusniger produces an antibacterial and antifungal compound ²⁰20. Tripathi C, Praveen V, Singh V, Bihari V. Production of antibacterial and antifungal metabolites by Streptomyces violaceusniger and media optimization studies for the maximum metabolite production. Med Chem Res. 2004;13(8-9):790-9..

The results obtained from the fractions’ MIC test show that the D fraction is the one with the

highest antimicrobial activity and concentration. Two chromatographic spots in this fraction were separated, this indicates a higher concentration of active metabolites with antimicrobial activity against Staphylococcus aureus strains.

The extract from Streptomyces sp. ERI-3 (compound isolated from Streptomyces sp) has been reported to produce inhibition of Staphylococcus aureus with a MIC of 0.25 mg/mL ¹⁹19. Arasu MV, Duraipandiyan V, Agastian P, Ignacimuthu S. In vitro antimicrobial activity of Streptomyces spp. ERI-3 isolated from Western Ghats rock soil (India). J Mycol Med. 2009;19(1):22-8.. SPG278 has a MIC of 256 ug/mL against Staphylococcus aureus (N6) and the extract of marine-related Streptomyces M10-77 has a MIC de 7.9 ug/mL ⁶6. León J, Aponte JJ, Rojas R, Cuadra D, Ayala N, Tomás G, et al. Estudio de actinomicetos marinos aislados de la costa central del Perú y su actividad antibacteriana frente a Staphylococcus aureus meticilina resistentes y Enterococcus faecalis vancomicina resistentes. Rev Peru Med Exp Salud Publica. 2011;28(2):237-46.; whereas, the D fraction of Streptomyces sp. 6E3 produced a MIC of 0.44 ug/mL against Staphylococcus aureus ATCC 43300 (MRSA).

The number of test repetitions is considered as one of the limitations of the study. It could not be determined whether the antimi crobial activity of the extract was in a compound within the cell or was excreted. Since several secondary metabolites may be present in each fraction of the extract, it is not clear whether the inhibition was due to the combined action of these metabolites or just by one of them.

In conclusion, Streptomyces sp. 6E3 showed increased antimicrobial activity against Staphylococcus aureus ATCC 33862 and MRSA strains.

References

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