Resazurin based rapid screening and Spectrophotometric analysis of potent antimicrobials from Streptomyces isolated in Nepal

Background The is a burning issue in the present medical development of novel classes of potent antibiotics. This study focuses on the robust assay of screening antimicrobials speeding up the antibiotics discovery process. Results Putative Streptomyces designated as LAf4 isolated from a soil sample of Lamjung, mid-hills of Nepal, screened from 141 putative Streptomyces isolates had the highest inhibitory effect among all the strains that showed inhibition potential against two of the ‘critical’ WHO prioritized pathogens including Klebsiella pneumoniae and Pseudomonas aeruginosa,. The results from Resazurin based assays were all verified using broth assays for antimicrobial studies. The proposed method was efficient, easy to observe and gave results a lot faster than the conventional antimicrobial study. Also, a novel spectrophotometric analysis method has been proposed to access the potency of thus screened antimicrobials. GCMS analysis of LAf4 extract indicated the presence of 2,3-butanediol, Naphthalene, and Tributyl phosphate as major constituents exhibiting antimicrobial properties whereas Pyrrolo (1, 2- a) pyrazine -1,4-dione, hexahydro-3-(2-methylpropyl)- which is an antioxidant and an anticancer agent was also recorded. Conclusion The present study validates and exhibits the time and cost effective resazurin based antimicrobial assay as the best one, for preliminary screening of antimicrobial producers compared to conventional disc/well diffusion assays since very small concentration of antimicrobials irrespective of their polarity can be detected unlike the latter methods.

assays were all verified using broth assays for antimicrobial studies. The proposed method was efficient, easy to observe and gave results a lot faster than the conventional antimicrobial study. Also, a novel spectrophotometric analysis method has been proposed to access the potency of thus screened antimicrobials. GCMS analysis of LAf4 extract indicated the presence of 2,3-butanediol, Naphthalene, and Tributyl phosphate as major constituents exhibiting antimicrobial properties whereas Pyrrolo (1, 2-a) pyrazine -1,4-dione, hexahydro-3-(2-methylpropyl)-which is an antioxidant and an anticancer agent was also recorded.
Conclusion The present study validates and exhibits the time and cost effective resazurin based antimicrobial assay as the best one, for preliminary screening of antimicrobial producers compared to conventional disc/well diffusion assays since very small concentration of antimicrobials irrespective of their polarity can be detected unlike the latter methods. 4

Background
The "Patent Cliff" (1) experienced by pharma-industries have not been able to bring adequately new antibiotics (2). The continued overuse and misuse of antibiotics that resulted in resistance (3) is further aggravated by emergence of multi-drug resistant (MDR) bugs (4) even to different last line of drugs (5) (6) and collective resistance to these arsenals (7). Historical serendipitous discoveries was the milestone in treating infections (8) followed by incremental modifications of the chemical scaffolds of the drugs based on mechanisms of inhibition after the 70's (9) that now appears to have exhausted and natural products have regained momentum (10), but with limited success (11).
The excessive time and cost parameters required to develop a drug, emphasized the search of time and cost effective protocols for preliminary screening of antimicrobials. For this purpose, Resazurin based microtitre-plate assay as described by Drummond and Waigh in 2000 was modified to develop a simple, rapid, sensitive and cost-effective in vitro antimicrobial assay. This method uses Resazurin, an indicator which detects the microbial growth based on its reduction potential by the reductases produced by viable cells. Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one) is a non-toxic, water soluble blue non-fluorescent dye which is converted into pink fluorescent resorufin (7-hydroxy-3H-phenoxazin-3-one) in the presence of viable cells with active metabolism (12). Reductases of viable cells reduce resazurin resulting in the formation of its highly fluorescent metabolic product resorufin. This reduction is proportional to the number of metabolically active cells present (13).
The present study aims to screen potent antimicrobials from Streptomyces extracts 5 isolated from various parts of Nepal based on Resazurin based antimicrobial assay and spectrophotometric analysis.

results and discussion
Screening of Streptomycetes and antimicrobial potential of cell culture extracts Putative Streptomyces were screened based on the typical morphology, chalky nonmucoid colonies ( Figure 1A and 1B) as recommended by Shirling and Gottlieb (14) with varying aerial and substrate mycelia colors (data not shown). Using ISP media for Streptomyces (15), a total of 141 colonieswere isolated. Out of these, 98 isolates were further pursued by screening out the look-alike isolates depending upon the culture and morphological characteristics ( Figure 1C and 1D) including aerial and substrate mycelia colors, septa formation, pattern of growth and so on.
Most of the concentrated extracts did not show antimicrobial potential against K. pneumonia and P. aeruginosa in agar disc diffusion test (data not shown) whereas some did show insignificant inhibitory zones.
Upon accessing the antimicrobial properties of the 14 selected colored culture extracts against the test pathogens, all 14 (Supplementary Table 1) showed potential inhibition against them to certain extent indicating that there could be compounds with antimicrobial potential in all of these extracts.

Resazurin antimicrobial assay and confirmation of isolate
A new spectrophotometric analysis strategy has been proposed to access antimicrobial potency of compounds in the mixture based on oxidation of resazurin as described in materials and methods. Depending upon the absorbance at 550 nm, 6 culture extracts of isolates LAf4, LAd1, LAe5 and HA5 showed relatively better antimicrobial potencies among the test strains. Extracts from LAf4 showed highest inhibitions against K. pneumoniae and P. aeruginosa. Also, LAf4, LAd1 LAe5 and HA5 showed similar or better antibacterial properties against these pathogens used at 20 mg/ml than Ampicillin and Kanamycin used at 50 mg/ml ( Figure 2). Among the tested, the extracts of the isolate KH8 showed the least inhibition potential against these test organisms.
The isolated putative Streptomycetes were confirmed by PCR amplification of the genomic DNA using StrepB/StrepE primer pairs designed specifically to identify Streptomycetes (16) which gave bands corresponding to 520 bp indicating that the isolates were Streptomyces sp. Furthermore, partial sequencing of the 16s rRNA genomic sequence of LAf4 and blast search among the Streptomycetesrevealed that the strain showed 87% identity out of 99% query cover with Streptomyces flavoviridis.

Gas chromatography-Mass spectrometry (GC-MS) analysis
The inhibitory potential of the LAf4 crude extract was further analyzed for the constituent compounds that could have potential antimicrobial properties by GCMS.
The most probable compounds indicated through the NIST library revealed that major constituents were antimicrobials (Table 1). discussion Streptomycetes are known to produce metabolites that act as antimicrobials and they also produce indole derivatives as antimicrobials (17). An ecologically diverse country Nepal, 49 th in the whole world (18), built through the rising of seabed from tectonic movement (19) could harbor different Streptomycetes including marine 7 which could produce potent secondary metabolites including antimicrobials. Thus, soil samples from 24 different parts of Nepal were collected for screening of Streptomyces species.
The isolates were cultured as described in materials and methods. The cell free culture media was further extracted using ethyl acetate as has been performed to extract metabolites produced by Streptomycetes (20) to obtain polar and non-polar secondary metabolites and preferred also because of its minimal toxicity to test organisms (21). Ethyl acetate was evaporated and the extract was concentrated as described to test its anti-microbial potential.
The insignificant inhibitory zones during agar diffusion antimicrobial susceptibility tests could indicate that there were some antimicrobial compounds whose concentrations were either low or, the insolubility in agar due to polarity (22) could have hindered the movement and efficacy of the potential antibacterial molecules in the medium.
Moreover, in the present study the search of novel and effective antimicrobials were pursued based on color forming isolates because colored indole derivatives, indirubin and isatin, have been reported as the major constituents of plant and microbial extracts with potent antimicrobial properties (23,24,25).
Since the world is in desperate need of novel antibiotics (26), even the extracts with small amount of antimicrobials should be considered as the source of new drug candidate as these could be novel and could function against the emerging resistance. Thus, effective and robust assay system should be developed where factors like polarity of the compound should not be stumbling block in antimicrobial assay. A robust and faster screening protocol for detecting antimicrobials is thus of dire need for which Resazurin based microbial viability assay could be modified for 8 antimicrobial screening.
During antimicrobial potency screening, the antioxidant property of the crude extract could be of concern. Since most antibiotics at present are known to produce reactive oxygen species (ROS) in killing the bacterial cells (27) (28)

Isolation
The air-dried soil samples were serially diluted in sterile distilled water and 100 μl of 10 -4 dilutions was plated on ISP2 and ISP4 among all the International Streptomyces Project (ISP) medias recommended by Shirling and Gottlieb (Shirling and Gottleib, 1996), using spread plating techniques. ISP2 is a general media with glucose as a common carbon source and yeast and malt extracts as other nutrient sources whereas ISP4 is more selective for Streptomyces screening. The agar plates were supplemented with 50µg/ml cycloheximide, an antifungal agent. The plates were incubated at 28°C for 2 weeks. The distinct strains were then selected based on their colony characteristics and subcultured on ISP2 agar plates for pure culture.

Secondary metabolites production
The putative Streptomycetes screened based on their colony characteristics were cultured in 50 ml ISP2 broths (pH-7) at 30°C for 7 days. The secondary metabolites containing media was then filtered out, separated using ethyl acetate in a separating funnel and concentrated using rotavapor at 38°C. All the crude extracts

Spectrophotometric readings
The color change from violet resazurin to pink resorufin in negative controls was considered the end point of the assay and the spectrophotometric reading was taken in microtiter plate reader at 550 nm.