Isolation, Identification, and Characterization of the Novel Antibacterial Agent Methoxyphenyl-Oxime from Streptomyces pratensis QUBC97 Isolate

Bacterial resistance to antibiotics is on the rise, both at the qualitative and quantitative levels. Searching for new antibiotics is an ongoing race in controlling pathogens, especially pathogens that have acquired antibiotic resistance. Our research may significantly contribute to finding new antibiotics. Our work has simplified and improved research tools and methods used for screening byproducts of Streptomyces spp. (or other sources such as plant extracts, fungi, Bacillus spp., and synthetic compound) for antibiotics or other bioactive agents. Our work may serve as a model for similar studies. Methoxyphenyl, oxime (MPO) may prove to be a therapeutic antibacterial agent that is effective against resistant bacteria such as the methicillin resistant staphylococcus aureus (MRSA). MPO may be prepared at commercial scale, synthesized, and modified to enhance its properties. Importance Isolation, Identification, and Characterization of the Novel Antibacterial Agent Methoxyphenyl-Oxime from Streptomyces pratensis QUBC97 Isolate Barghouthi SA*1, Ayyad I1, Ayesh M1, and Abu-Lafi S2 1Medical Laboratory Sciences, Faculty of Health Professions, Jerusalem, Palestine 2Faculty of Pharmacy, Al-Quds University, Jerusalem, Palestine *Corresponding author: Barghouthi SA, Medical Laboratory Sciences, Faculty of Health Professions, Jerusalem, Palestine, Fax: +972-2-2791234, Tel: +972-599-379561, E-mail: bargsam@yahoo.com


Introduction
Identification of Streptomyces species and the bioactive compound(s) they produce are complex and problematic [1][2][3][4]. Major obstacles in identifying Streptomyces species include horizontal gene transfer, erroneous reporting of species [5], recombination, chromosome rearrangement that may take place in Streptomyces [6,7], and replacing the scientific species name with a number Volume 1 | Issue 1 We suggest the creation of a "Hybrid Bank of Taxonomy" connecting morphological features to DNA sequences such as 16S and/or other sequences; i.e. Streptomyces grouped based on 16S similarity must then be distinguished based on morphological, molecular features, signature protein(s), DNA "tags", and/or distinguishable character of a species may provide such a simple system of species identification [this work, [3][4][5][8][9][10]. Taddei et al. [3] applied morphological and biochemical tests to differentiate between 71 isolates obtained from Venezuelan soils. The tests failed to confirm or rule out identity [3]. Ju et al. identified gene clusters of phosphonic acid metabolism among 10,000 actinomycetes genomes. They classified gene clusters into 64 clusters encompassing 278 strains. These clusters may represent important tags for such strains allowing their separation from other actinomycetes. They also identified eleven undescribed compounds one of which is phosphonocystoximate [4]. Gao and Gupta discussed "The Molecular Signatures" for Actinobacteria. The concatenated protein sequences represent molecular signature proteins placing sequenced Streptomyces spp. in a single cluster [8]. Rong et al. utilized five concatenated DNA sequences to construct a phylogenetic tree that was different from the 16S-phylogenetic tree of the same isolates [5]. Shirling and Gottlibe indicated that their goal was to identify stable taxonomical properties of Streptomyces spp. [9]. Seventy years ago, Waksman and Lechevalier published their manual for Streptomyces classification and identification [10].

Journal of Antibiotics Research
Such a bank of information will allow systematic analyses of all species and their by-products if we are to fight multidrug resistant microbes and cancer. *Total identity (Ti) = A × B; exact identity =100%, if A = 50% and B = 90%, then Ti = 45% Table 1: BLAST results with genome of Streptomyces griseus subsp. Griseu. Accession |AP009493.1| showing a huge range of variation among a short list of selected species of the genus Streptomyces indicating the high diversity of the genus (e.g. Streptomyces sp. Xxxx) which increases uncertainty of species identity. Discrepancies between 16S based phylogrouping and multilocus based phylogrouping suggest that accurate species identification is difficult [5]. Sequences of 16S of Streptomyces phylogroup pratensis are identical for eight species in the S. griseus clade. Such diverse identity (calculated by multiplying BLAST sequence coverage by its percent BLAST identity) showing genomic variations between species of the genus Streptomyces to range from 20.4 to 100% in this example (Table 1). A practical system to reclassify Streptomyces species may be necessary.
In this work, we report the isolation of the antibacterial agent methoxyphenyl-oxime as identified by MS analysis from a Streptomyces isolate QUBC97 which was closely related to Streptomyces phylogroup pratensis as suggested by phenotypic, multilocus, and DNA sequence analyses.

CCG culture medium and isolation of Streptomyces
Unrelated soil samples (200-300 g) were collected from a depth of 5cm in fresh plastic bags and stored in wooden drawers at room temperature. CCG selective agar medium (0.1% casamino acids proteolytic hydrolysate, Sigma Chemicals; 0.1% trisodium citrate; 2% agar made in Jericho® bottled water as a source of trace elements, pH ~7.8 before autoclaving;). Autoclaved media (broth or agar) were cooled to ~50 ºC; autoclaved glycerol (50% v/v made in water) was aseptically added to a final concentration of 0.5% v/v. Filter sterilized cycloheximide at 100µg/ml was added to CCG medium to make CCCG medium which was used occasionally to control contaminating fungi. CCG cultures were used to evaluate bioactivities of Streptomyces spp. Nutrient agar from BD Difco, USA was used in disc diffusion tests and the initial isolation of isolate QUBC97 from a commercial cacao sample, the isolate was transferred to CCG medium.

Characterization and identification of the isolate QUBC97
Streptomyces isolate color was observed on ISP4 (BD Difco) [9]. Microscopic examination at 4x, 10x, and 45x objectives of isolate QUBC97, documented with Exilim camera, Casio, Japan). The guide for identification of Streptomyces sp. was consulted [10].
Several loci, 16S, superoxide dismutase (SOX), and amylase were employed in the identification of QUBC97 in addition to morphological features. The loci, primers, and PCR product size are detailed (Table 2) [11,12]. Sequenced amplicons and multilocus analyses were employed to include or exclude QUBC97 from lists generated by primer nucleotide-BLAST.
Solvent systems used to develop the TLC strips were: The top layer of n-butanol equilibrated with 1.7% ammonium acetate (nBA) [15]. Other solvent systems were prepared according to HEMW system; Hexane, Ethyl acetate, Methanol, and Water [16]. Selected amplicons were extracted using NeucleoTrap, Mecherey Nagel, Germany and sequenced (Bethlehem University, Bethlehem, Palestine) then analysed by BLAST nucleotide alignment.
Three simple screening tests were devised and applied in antibiotic evaluation.

Antibacterial bioassay tests
Agar plug diffusion test: Four-week old Streptomyces CCG agar plates (28 °C) were used to obtain long agar plugs using a sterile scalpel blade or the wide end of Pasteur pipette kept in 70% alcohol, drained, flamed, cooled, and used to cut agar plugs near growth areas. Agar plugs were placed on lawns of target bacteria/yeast (Bacillus atrophaeus QUBC16, baker's yeast Sacchromyces cerevisoae, or other bacterial isolates) prepared on nutrient agar (NA) plates [13,14]. After overnight incubation at 30 °C, zones of inhibition were observed. Repetition was done to confirm results. n-butanol extraction: One ml aliquot was collected from 50-ml liquid CCG culture grown in 150 ml-Erlenmeyer flasks; shaking at 120 strokes/min at 28 °C for indicated time, cleared by centrifugation. The supernatant was vigorously mixed with 0.3 ml n-butanol and briefly centrifuged. Ten µl of butanol top layer were placed on 3-mm sterile paper discs, air dried, and placed on bacterial lawns prepared on NA plates, incubated at 30 °C and inspected after overnight incubation for zones of inhibition.
This method was used to study the kinetics of antibacterial production; Three filter paper discs were impregnated each with 10 µl n-butanol extracted daily for 7 days, allowed to air-dry, and placed on the surface of NA coated with Bacillus atrophaeus QUBC16 [13]. Diameters of zones of inhibition were measured to reflect productivity.
Large scale extraction of antimicrobial activity of Streptomyces QUBC97, two 750 ml CCG medium each placed in 2-liter flasks with shaking (120 Strokes /min) for 2 weeks at 28 °C, centrifuged in 30-ml tubes at 25,000 rpm for 15 min, supernatant was pooled in a separatory funnel, mixed vigorously with n-butanol (4:1 v/v) respectively, allowed to settle overnight, and the upper layer was collected. A second extraction was performed similarly. The two n-butanol fractions were pooled and evaluated by the disc diffusion method. The extract labelled as crude n-butanol extract (cnBE). Using rotary evaporator at 50 °C, cnBE was concentrated to near dryness.

Evaluation of antimicrobial agent mobility by thin layer chromatography (TLC):
Ten µl of cnBE from broth or agar plates were spotted on the base line of silica thin layer chromatography plate strips (1 × 8 cm), allowed to air-dry and developed in different solvents. Dried TLC strips were visualized under UV light, and/or placed face down on lawns of target organism on NA plates, after overnight incubation at 30°C plates were examined for presence of zones of growth inhibition.

Journal of Antibiotics Research
Putative identification of the aerobic colonies showing fine microscopic filamentous mycelia, and compact colonies with or without powdery spores were essential features that putatively identified Streptomyces colonies. ISP4 and CCG cultures were used for further macroscopic and chemical characterization ( Figure 1). Concentrated cnBE (n-butanol extraction section) was dissolved in water and subjected to preparative HPLC purification. Four peaks wear eluted in water, only one peak showed antibacterial activity as evaluated by disc diffusion method. The two positive fractions (50ml each) were pooled and re-extracted with 25 ml n-butanol from the aqueous HPLC column fractions and concentrated by rotary evaporation at 50 °C. The dried material was re-suspended in 1 ml autoclaved pure water for analysis by mass-spectroscopy for putative identification.
Ten µl-discs were air-dried (n-butanol extraction section), diffusion method was used against a number of bacterial species: Staphylococcus epidermidis, several S. aureus isolates, Streptococcus viridians, S. pyogenes, S. aglactiae, Bacillus atrophaeus, Escherichia coli (several isolates), E. coli HB101 laboratory strain, and several isolates of Pseudomonas aeruginosa. The cnBE extract was tested against three clinical Methicillin Resistant S. aureus (MRSA) and baker's yeast as well.

Isolation and maintenance of Streptomyces species
Tens of Streptomyces spp. were simply obtained from soil samples on CCG medium. QUBC97 was initially isolated on nutrient agar and maintained on CCG medium. Microscopy allowed quick subculturing of microscopic colonies that were identified by direct microscopic (4× and 10× objectives) examination of CCG plates.
Screening several isolates with the Agar Plug Diffusion assay, QUBC97 was selected for further investigation. Colonies of isolate QUBC97 showed white areal powdery mycelia and yellow growth on both CCG Figure (Figure 2), antibiotic diffusion agar plugs were used as in filter paper disc sensitivity tests. Regardless of shape of agar plugs, agar plugs were placed on Bacillus atrophaeus QUBC16 lawns prepared on nutrient agar and incubated at 30 °C. Figure 3 shows QUBC97 and two other isolates that produced anti-bacillus agents. QUBC97 was selected for further investigation. Figure 4 shows zones of inhibition generated by crude n-butanol extract ( (Figure 3), Escherichia coli DH5α, clinical P. aeruginosa 7, and baker's yeast.

QUBC97 activity against clinical isolates
Longitudinal sampling of liquid culture medium every 24 hours for seven days followed by immediate extraction with n-butanol, and disc diffusion assay against B. atrophaeus QUBC16 revealed varying zones of inhibition. Diameter of zone of inhibition was viewed as a direct reflection of the concentration of antibacterial agent. For each time point 3 readings were obtained. Diameters of zones of inhibition were measured after 24 h incubation at 28 °C ( Figure 5).

Kinetics of antibiotic production
Volume 1 | Issue 1 Journal of Antibiotics Research The production of the antibiotic appeared one day post incubation and increased with time and reached its maximum level after 6 days of incubation as reflected by the 21-mm zone of inhibition.
The recovered n-butanol extract from agar plates of QUBC97 was analyzed by thin layer chromatography on silica gel plates (TLC). The results showed that two compounds can be separated into two bioactive spots when TLC was developed in n-butanol saturated with 1.7% ammonium acetate (nBA); Rf-values were 0.62 and 0.85 ( Figure 6).

TLC analysis of butanol extract
In another solvent (water: methanol; 1:1 v/v), Rf was 0.17 and another product appearing as a streak along the TLC strip as indicated by the zone of inhibition along the TLC plate ( Figure 6). When extracts were obtained from liquid QUBC97-cultures, only one (fresh FCm or old OCm) product could be extracted with nbutanol depending on the age of culture. Therefore, liquid media provided a simple method to obtaining one or the other product for further analysis. FCm and OCm products prepared separately, when co-spotted on the same TLC plate they showed different Rf -values ( Figure 7).  MS analysis of the HPLC purified sample suggested the chemical formula C 8 H 9 NO 2 , identified with 80% probability as methoxyphenyl-oxime ( Figure 8A). Probability of being 4-ethylbenzoic acid, 2-butyl ester probability was 6.84%; and 4-ethylbenzoic acid, cyclopentyl ester 6.04%. The MS spectrum of the compound was different from that of ethanone, 1-phnyl-, oxime. The n-butanol Chemical nature of the n-butanol extracted antibacterial agent extract remained bioactive after 3 years of preparation and storage at room temperature. Changing the pH of agar plugs by soaking in sodium hydroxide or hydrochloric acid appeared to increase the zone of inhibition ( Figure 8B).
Annex Publishers | www.annexpublishers.com   Figure 9 shows PCR amplicons of superoxide dismutase (SOX), amylase amplicon, and a number of 16S amplicons, each amplicon was generated from QUBC97 with Streptomyces general primers. BLAST analysis of 502-nucleotide sequence of the 16S amplicon (Table 3) matched 17 Streptomyces spp. with 100% coverage and 99% identity ( Table 4) including most of the S. pratensis phylogroup [5]. BLAST results of superoxide dismutase (SOX: 329 b) sequence (Table 3) returned 190 matches with 99% identity. Common species that matched both SOX and 16S identified QUBC97 as Streptomyces pratensis ATCC 33331, S. microflavus, or S. fulvissimus. Since QUBC97 produced positive amylase PCR reaction (386bp), S. microflavus was eliminated because it produced a negative amylase BLAST result. It also was negative when BLAST-analyzed with amylase primers (Table 2). S. fulvissimus and S. pratensis shared amylase amplicon and could not be excluded. However, both S. microflavus and S. fulvissimus unlike QUBC97 do not produce diffusible pigments on organic media [10]. Analyses suggested that QUBC97 was most likely related to S. pratensis phylogroup [5].  This work contributed the formulation and application of the new Citrate-Casamino-Glycerol (CCG) medium as a medium for physiological manipulations and by-product analyses. Tens of confirmed Streptomyces isolates were obtained [11][12][13] in the absence of cyclohexamide, nalidixic acid, or nystatine [18]. The selectivity of GCC medium may be related to its alkaline pH (~7.8 before autoclaving) and poor nutritional content. The transparency of the medium allowed direct microscopic examination and enabled putative identification of Streptomyces (Figure 1). Simple media are preferred for assaying antibiotic production; glycerol is used by several Streptomyces spp. [19,20]. Agar Plugs used in antibiotic screening were cut from three-week old Streptomyces CCG agar cultures ( Figure 3) and were effectively used in presumptive evaluation of antimicrobial activity.

Discussion
Integration of molecular and morphological methods appeared to simplify Streptomyces species identification which is not a simple task [5]. Superoxide dismutase (SOX) and 16S sequencing, amylase PCR and morphological characteristics identified Streptomyces QUBC97 as a member of the Streptomyces pratensis phylogroup, a results that is in agreement with Rong et al report [5]. QUBC97 matched 7 of the species placed in the S. pratensis phylogroup based on 16S sequences and matched the strains S. pratensis ATCC33331 and S. griseus NBRC 13350 of the same 16S phylogroup [5]. Yet genome BLAST alignment of Streptomyces flavogriseus strain ATCC 33331 (Streptomyces flavogriseus IAF-45-CD =ATCC 33331 recently reclassified as S. pratensis ATCC33331) [1], revealed S. griseus NBRC 13350 as the closest match. Only 58% genome coverage and 88% identity (Ti = 51%) whereas Ti was near 100% based on 16S matching. Although QUBC97 was closely related to Streptomyces pratensis ATCC 33331. Despite some discrepancies that separated QUBC97 from S. pratensis ch24, ATCC 33331, and other S. pratensis phylogroup species and strains; we identified the isolate as S. pratensis QUBC97.
This report is the first to show antibacterial activity of MPO from Streptomyces pratensis QUBC97, related oxime containing compounds other than MPO have been recovered from Streptomyces cultures and other sources [21][22][23][24]. Several synthetic oximes with antifungal properties have been reported [25]. Maize plant cytochrome P450 forms oximes from their aminoacids; phenylacetaldoxime and indole-3-acetaldoxime [26].

Journal of Antibiotics Research
Chipps et al. reported on the cytotoxicity of MPO extracted as an aqueous component of bitter melon seed [27]. MPO is present as 4.34% fraction in ethanol extract of the plant Sedum pallidum; the crude extract showed antibacterial activity [28].
It is not clear how methoxyphenyl-oximes and other oximes behave as antibiotics. We have neither investigated whether MPO was bactericidal or bacteriostatic nor we have investigated its mechanism of action. Methoxyphenyl-oxime glycosides named Uzmaq-A and Uzmaq-B isolated from Aspergillus flavus AF612 are biosurfactants [29].

Conclusion and Recommendation
However, since MPO was not effective against some bacterial isolates and Baker's yeast we assumed it was not toxic and may have specific bacterial (prokaryotic target). MPO was effective against all three tested clinical isolates of Methicillin Resistant Staphylococcus aureus (MRSA) indicating it is separable from β-lactam antibiotics. Although MPO was effective against some Gram negative bacteria, others were resistant; these were E. coli DH5α but not HB101, Pseudomonas aeruginosa 1 but not P. aeruginosa 7 ( Figure 4) suggesting that MPO may be a true specific antibacterial agent that controlled some bacteria but not others.
The appearance of another active antibacterial agent in old culture broth (OCm) and disappearance of the fresh methoxyphenyloxime (FCm) was noticed. Both shared similarities; split peak light absorbance at 426/428 and 242 nm and anti-bacillus bioactivity. However, their mobility differed on TLC developed in nBA when co-spotted or when spotted individually, suggesting that one molecule was likely metabolized further to acquire a different form with different Rf value. TLC mobility of the compound in different solvents was consistent with MPO structure in terms of molecular polarity. When agar plugs were acid-or alkali-treated, increased zones of inhibition were observed, this enhancement may be due to increased agent solubility. Apparently, the acid/alkaline treatment did not cause degradation of the antibacterial function ( Figure 8B). These characteristics of soluble diffusible yellow pigment that was extractable in n-butanol, with indicated Rf values, and light absorption at 426/428 and 442 nm may represent characteristic tags to S. pratensis QUBC97 or similar strains. This QUB97 tag is similar to the intracellular or extracellular blue pigment tag produced by S. coelicolor A3(2) at different pH ranges described by Bystrykh et al. [30].
We recommend that researchers provide better description of their Streptomyces isolates since 16S DNA sequence must be coupled with other molecular, morphological, biochemical characteristics, or tags. Representative photos of colony color and morphology may aid others to compare strains and isolates. The TLC bioassay system applied in this study (Figures 6 and 7) is a powerful tool towards preliminary characterization of the antibiotic agent, small scale purification, testing fractionated products from HPLC, extracts, or other preparation. It may provide an excellent means for comparative antibiotic studies.
Thanks to Al-Quds University and Dr. Ibrahim Abbassi, Al-Quds University for his suggestions and support. This work was presented as Master's Thesis [31].